U.S. patent number 10,407,673 [Application Number 16/011,622] was granted by the patent office on 2019-09-10 for methods for glycoprotein remodeling using endoglycosidase mutants.
This patent grant is currently assigned to Academia Sinica, CHO Pharma Inc.. The grantee listed for this patent is Academia Sinica, CHO Pharma Inc.. Invention is credited to Li-Tzu Chen, Ting Cheng, Lin-Ya Huang, Nan-Horng Lin, Sachin S Shivatare, Chi-Huey Wong, Chung-Yi Wu.
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United States Patent |
10,407,673 |
Lin , et al. |
September 10, 2019 |
Methods for glycoprotein remodeling using endoglycosidase
mutants
Abstract
A mutant of EndoS2 includes one or more mutations in the
sequence of a wild-type EndoS2 (SEQ ID NO:1), wherein the one or
more mutations are in a peptide region located within residues
133-143, residues 177-182, residues 184-189, residues 221-231,
and/or residues 227-237, wherein the mutant of EndoS2 has a low
hydrolyzing activity and a high tranglycosylation activity, as
compared to those of the wild-type EndoS2. A method for preparing
an engineered glycoprotein using the mutant of EndoS2 includes
coupling an activated oligosaccharide to a glycoprotein acceptor.
The activated oligosaccharide is a glycan oxazoline.
Inventors: |
Lin; Nan-Horng (Vernon Hills,
IL), Huang; Lin-Ya (New Taipei, TW), Shivatare;
Sachin S (Taipei, TW), Chen; Li-Tzu (Taipei,
TW), Wong; Chi-Huey (Taipei, TW), Wu;
Chung-Yi (New Taipei, TW), Cheng; Ting (Keelung,
TW) |
Applicant: |
Name |
City |
State |
Country |
Type |
CHO Pharma Inc.
Academia Sinica |
Taipei
Taipei |
N/A
N/A |
TW
TW |
|
|
Assignee: |
CHO Pharma Inc. (Taipei,
TW)
Academia Sinica (Taipei, TW)
|
Family
ID: |
61241785 |
Appl.
No.: |
16/011,622 |
Filed: |
June 18, 2018 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180298361 A1 |
Oct 18, 2018 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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15684897 |
Aug 23, 2017 |
10000747 |
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62378806 |
Aug 24, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P
21/005 (20130101); A61P 35/00 (20180101); C12Y
302/01096 (20130101); C07K 16/2887 (20130101); A61P
35/02 (20180101); C12N 9/2434 (20130101); C07K
2317/41 (20130101); C07K 2317/732 (20130101); C07K
2317/24 (20130101); C07K 2319/21 (20130101); C07K
2317/72 (20130101) |
Current International
Class: |
C12N
9/42 (20060101); C12P 21/00 (20060101); C07K
16/28 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Sjogren et al. (Biochem. J. (2013) 455, 107-118). cited by examiner
.
Li et al. (The Journal of Biological Chemistry vol. 291, No. 32,
pp. 16508-16518, Aug. 5, 2016). cited by examiner.
|
Primary Examiner: Noakes; Suzanne M
Assistant Examiner: Lee; Jae W
Attorney, Agent or Firm: Liang Legal Group, PLLC
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This claims the priority of U.S. Patent Application No. 62/378,806,
filed on Aug. 24, 2016, the disclosure of which is incorporated by
reference in its entirety.
Claims
What is claimed is:
1. A method for preparing an engineered glycoprotein using a mutant
endoglycosidase S2 (EndoS2), comprising coupling an activated
oligosaccharide to a glycoprotein acceptor, wherein the mutant
EndoS2 comprises one or more mutations in the sequence of a
wild-type EndoS2 as set forth in SEQ ID NO:1, wherein the one or
more mutations are in a peptide region located within residues
133-143, residues 177-178 residue 182, residues 187-189, residues
221-231, and residues 232-237, wherein the mutant of EndoS2 has a
hydrolyzing activity lower than that of the wild-type EndoS2 and
has a tranglycosylation activity higher than that of the wild-type
EndoS2, and wherein the mutation at residue 182 is D I82Q.
2. The method according to claim 1, wherein the one or more
mutations are at residues T138, D226, T227, and/or T228.
3. The method according to claim 1, wherein the one or more
mutations are selected from the group consisting of T138D, T138E,
T138F, T138H, T138K, T138L, T138M, T138N, T138Q, T138R, T138V,
T138W, D226Q, T227Q, and T228Q.
4. The method according to claim 1, wherein the mutant comprises
the sequence of SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9,
SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID
NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO. 2, SEQ
ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5.
5. The method according to claim 1, wherein the activated
oligosaccharide is a glycan oxazoline.
6. The method according to claim 5, wherein the glycan oxazoline
comprises an N-glycan having the following formula: ##STR00006##
wherein R.sup.1 is --H or N-acetyl glucosamine attached via a
.beta.-1,4 linkage, and R.sup.2 and R.sup.3 are same or different
and are independently selected from the group consisting of:
mannosyl-(Man), di-mannosyl-(Man.sub.2), tri-mannosyl-(Man.sub.3),
biantennary penta-mannosyl-(Man.sub.2-(Man.sub.2)Man), biantennary
tetra-mannosyl-(Man.sub.2-(Man)Man), biantennary
tri-mannosyl-(Man-(Man)Man),
N-acetylglucosamine-mannosyl-(GlcNac-Man),
galactose-N-acetylglucosamine-mannosyl-(Gal-GlcNAc-Man),
N-acetylneuraminic
acid-galactose-N-acetylglucosamine-mannosyl-(Nur5Ac-Gal-GlcNAc-Man),
biantennary di-N-acetylglucosamine-mannosyl(GlcNAc).sub.2-Man),
biantennary
di-(galactose-N-acetylglucosamine)-mannosyl-((Gal-GlcNAc).sub.2-Man),
biantennary di-(N-acetyl-neuraminic
acid-galactose-N-acetylglucosamine)-mannosyl-((Neu5Ac-Gal-GlcNAc).sub.2-M-
an),
galactose-(fucosyl-N-acetylglucosamine)-mannosyl-(Gal-(Fuc-GlcNAc)-Ma-
n), N-acetylneuraminic
acid-galactose-(fucosyl-N-acetylglucosamine)-mannosyl-((Neu5Ac-Gal-(Fuc-G-
lcNAc)-Man),
fucosyl-galactose-N-acetylglucosamine-mannosyl-fucosyl-galactose-(fucosyl-
-N-acetylglucosamine)-mannosyl-(Fuc-Gal-GlcNAc-Man), N-neuraminic
acid-(fucose-galactose)-N-acetylglucosamine-mannosyl-(Nur5Ac-(Fuc-Gal)-Gl-
cNAc-Man),
fucose-galactose-(fucose-N-acetylglucosamine)-mannosyl-(Fuc-Gal-
-(Fuc-GlcNAc)-Man), N-neuraminic
acid-(fucose-galactose)-(fucose-N-acetylglucosamine)-mannosyl-(Neu5Ac-(Fu-
c-Gal)-(Fuc-GlcNAc)-Man),
(galactose-N-acetylglucosamine).sub.n-galactose-N-acetylglucosamine-manno-
syl-(Gal-GlcNAc).sub.n-Gal-GlcNAc-Man),
(galactose-N-acetylglucosamine).sub.n-galactose-(fucose-N-acetylglucosami-
ne)-mannosyl-((Gal-GlcNAc).sub.n-Gal-(Fuc-GlcNAc)-Man),
N-acetylneuraminic
acid-(galactose-N-acetylglucosamine).sub.n-galactose-N-acetylglucosamine)-
-mannosyl-(Neu5Ac-(Gal-GlcNAc).sub.n-Gal-GlcNAc-Man), and
N-acetylneuraminic
acid-(galactose-N-acetylglucosamine).sub.n-galactose-(fucose-N-acetylgluc-
osamine)-mannosyl-(Neu5Ac-(Gal-GlcNAc).sub.n-Gal-(Fuc-GlcNAc)-Man),
wherein n is an integer from 1 to 3.
7. The method according to claim 1, wherein the glycoprotein
acceptor contains a GlcNAc monosaccharide.
8. The method according to claim 1, wherein the glycoprotein
acceptor is a non-fucosylated GlcNAc-acceptor.
9. The method according to claim 1, wherein the glycoprotein
acceptor is a glycopeptide, a glycoprotein, an antibody or a
fragment thereof.
10. The method according to claim 1, wherein the glycoprotein
acceptor is a core fucosylated or non-fucosylated GlcNAC-IgG
acceptor or a fragment thereof.
11. The method according to claim 1, wherein the GlcNAC-IgG
acceptor is derived from a monoclonal antibody selected from the
group consisting of cetuximab, rituximab, muromonab-CD3, abciximab,
daclizumab, basiliximab, palivizumab, infliximab, trastuzumab,
gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan,
adalimumab, omalizumab, tositumomab, 1-131 tositumomab, efalizumab,
bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept,
IGN101, volociximab, Anti-CD80 mAb, Anti-CD23 mAb, CAT-3888,
CDP-791, eraptuzumab, MDX-010, MDX-060, MDX-070, matuzumab,
CP-675,206, CAL, SGN-30, zanolimumab, adecatumumab, oregovomab,
nimotuzumab, ABT-874, denosumab, AM 108, AMG 714, fontolizumab,
daclizumab, golimumab, CNTO 1275, ocrelizumab, HuMax-CD20,
belimumab, epratuzumab, MLN1202, visilizumab, tocilizumab,
ocrerlizumab, certolizumab pegol, eculizumab, pexelizumab,
abciximab, ranibizimumab.
12. A method for treating cancer, comprising administering to a
subject in need thereof an effective amount of a glycoprotein
prepared by the method of claim 1.
Description
BACKGROUND OF INVENTION
Field of the Invention
The present invention relates to selected mutants of
endoglycosidase S2 (EndoS2) from Streptococcus Pyogenes that
display improved transglycosylation activities and reduced
hydrolyzing activities for the synthesis of glycoproteins or
glycopeptides carrying a broad range of well-defined N-glycans of
high mannose, hybrid and complex types. In particular, one or more
embodiments of present invention also relate to use of EndoS2
mutants for efficient glycan remodeling of therapeutic antibodies
to form homogenous glycan compositions at Fc-domain for improvement
of their effector functions.
Background Art
Since the approval of the first therapeutic monoclonal antibody
therapy in 1986, the commercial pipeline of this class of
biopharmaceutical products have become most robust and dynamic (1).
As of early 2015, a total of forty-seven monoclonal antibody
products have been approved in the U.S. or Europe for the
treatments of a variety of diseases, including cancer, autoimmune
and infectious diseases. If it continues at the current approval
rate, approximately 70 monoclonal antibody products will hit the
market by 2020, and account for worldwide sales of nearly $125
billion (2). For monoclonal antibody therapeutics that depend on
Fc-mediated effector functions for their clinical activities, the
compositions of N-glycans at the Fc domains have been shown to be
critical for safety or efficacy (3). Diverse glycosylation states
have also been implicated to influence the pharmacodynamic and
pharmacokinetic properties, while other Fc glycan structural
elements may be involved in adverse immune reactions. However, the
way to control the Fc-glycosylation remains challenging.
A typical IgG consists of two antigen-binding fragments (Fabs),
which are connected via a flexible region to a constant region
(Fc). The Fab domains are responsible for antigen recognition while
the N-glycan at Asn297 of Fc domain interact with respective
Fc.gamma. receptors (such as Fc.gamma.RIIIa and Fc.gamma.RIIb) on
effector cells and Clq component of the complements that activate
the effector functions, including antibody-dependent cellular
cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)
(5-7). Almost all therapeutic antibodies are N-glycosylated on each
of the homodimeric Fc domains at the conserved asparagine residue
(N297). These N-linked glycans result in more than 30 different
glycoforms, and are typical biantennary complex type with
considerable structural heterogeneity, in which the core
heptasaccharide can be differentially decorated with core fucose
(Fuc), bisecting N-acetylglucosamine (GlcNAc), terminal galactose
(Gal), and terminal sialic acid (Sia) (8-9). The composition of
N-glycans could influence the Fc domain conformation, therefore,
modulating the antibody's stability, pharmacokinetic profile,
immunogenicity, effector functions, antibody-mediated inflammation,
and complement activation (10). For example, the absence of the
core fucose, as well as the attachment of a bisecting GlcNAc
moiety, dramatically enhances the affinity of antibody for the
Fc.gamma.IIIa receptor (Fc.gamma.RIIIa) on effector cells,
resulting in better elimination of the target (10-11). In addition,
the terminal a-2,6-sialylated glycan, which is a minor component of
antibodies and the intravenous immunoglobulin (IVIG), is an
optimized structure that enhances the anti-inflammatory properties
(12-13).
N-glycosylation is one of the most complex post-translational
modifications that often result in a remarkable heterogeneity of
glycan structures including high mannose, hybrid and complex types,
depending on the recombinant expression system (14-15).
Commercially available therapeutic antibodies typically exist as
mixtures of glycoforms that are not optimal for their respective
therapeutic activities. Recently, glycoengineering has gathered a
huge attention to control Fc glycosylation for improving efficacy.
One of the most common methods is the in vivo engineering of
synthetic pathways on the expression host. However, the glycoforms
generated by this method are limited, and total control over the
desired glycoform cannot be achieved. An alternative way to address
the glycosylation heterogeneity is endoglycosidases [endo-N-acetyl
glucosaminidase (ENGase)] with or without fucosidase mediated
trimming off all the heterogeneous N-glycans to leave only the
first GlcNAc or Fuc-GlcNAc at the glycosylation site of IgGs and
then a well-defined activated glycan in the form of oxazoline can
be transferred back on GlcNAc acceptor to form a natural .beta.-1,4
linkage (16).
Endoglycosidases, EndoS and EndoS2, are a family 18 glycoside
hydrolase (GH) from the human pathogen Streptococcus pyogenes and
have recently become the point of attention for glycoengineering of
therapeutic antibodies (17-18). Despite their mere 37% sequence
identity, both EndoS and EndoS2 catalyze the hydrolysis of the
.beta.-1, 4 linkage between the two N-acetylglucosamines (GlcNAcs)
in the core of the N-linked glycan of human IgG. Additionally, both
enzymes remove complex type glycans at IgG Fc domain. However,
EndoS2 can hydrolyze hybrid and oligomannose structures to a
greater extent, as compared with EndoS (19). Moreover, in the
presence of sugar-oxazolines as substrates, some endoglycosidases
of GH18 and GH85 turn into glycosynthases to catalyze chitobiose
linkage. However, the intrinsic hydrolyitic activity of these
glycosynthase enzymes reduces the overall yields.
Further improvement on enzyme activity leads to the development of
endoglycosidase mutants, including EndoS mutants D233Q and D233A
(16). This revelaed important motifs in the active site of ENGase:
D-X-E in GH18. This motif supports the catalysis mechanism of
ENGase, which uses a double-displacement reaction with neighboring
group participation. In this mechanism, the 2-acetamide group of
the GlcNAc acts as a nucleophile to replace the leaving group at
the anomeric center, with the formation of an oxazolinium ion
intermediate promoted by the carboxylate of Asp. The catalytic
residue, glutamate, acted as a general acid/base that protonates
the leaving aglycan group and deprotonates the nucleophilic
H.sub.2O leading to the hydrolysis of the oxazolinium ion
intermediate to form a hydrolytic product. The carboxylate of
aspartate, second residues on the N-terminal sides of E, was
proposed to promote and stabilize the formation of the oxazolinium
ion intermediate. The D233Q mutation of EndoS was demonstrated to
improve transglycosylation and diminish hydrolysis activity.
The above-described EndoS mutants are the best glycosynthase known
so far. They have a great potential for the synthesis of
homogeneous antibodies. However, a large amount of enzymes is
usually require to achieve complete conversion of the starting
material into a product. Therefore, the large scale production of
enzymes and the downstream purification to remove residual enzyme
content after reaction become tedious and labor intensive, which
further attribute to the high cost of the overall glycoenginnering
process. In addition, the transglycosylation activity of EndoS
mutants is limited particularly to symmetric bi-antennary complex
types glycoforms, but not towards a wide range of high mannose,
hybrid and tri- and tetra-antennary complex type glycans having
additional native modifications such as .alpha.-1,3-fucose on
GlcNAc, .alpha.-1,2-fucose on Gal, extended poly LacNAc motifs, and
asymmetric sialylated antennae at the termini.
Because of its potential to hydrolyze a broad range of IgG glycans,
EndoS2 generated a huge interest in the IgG glycoengineering field.
Based on the sequence alignment results of EndoS and EndoS2, it has
been speculated that the site D184 of EndoS2 is identical to the
site D233 of EndoS. Therefore, it is expected that the mutants
format this site D184 of Endo S2 might enhance transglycosylation
activity and diminish hydrolytic activity of EndoS2. In light of
above-mentioned prior art, it would be advantageous to improve the
glycosynthase activity of EndoS2 by site directed mutagenesis near
the active site of the wild-type EndoS2. Provided herein are EndoS2
mutants having excellent glycosynthase activity and decreased
hydrolyitic activity. More importantly, novel EndoS2 mutants of
this invention offered broad substrate range, and able to transfer
high mannose, hybrid and bi- and tri-antennary complex type
N-glycans in the form activated glycan oxazolines. EndoS2 mutants
of this invention facilitate production of diversely glycosylated
homogeneous antibodies, particularly of those fully sialylated
multi-antennary glycoforms that expected to gain anti-inflammatory
activities, for biophysical and structural studies.
SUMMARY OF INVENTION
Embodiments of the present invention relates to selected mutants of
EndoS2 that show reduced hydrolyzing activities and excellent
transglycosylation activities against a broad range of N-glycans of
high mannose, hybrid and complex types. These EndoS2 mutants may be
used to prepare homogeneously glycosylated glycopeptides,
glycoproteins, and therapeutic antibodies or Fc fragments thereof.
Embodiments of the present invention allow for efficient glycan
remodeling of therapeutic antibodies and Fc fragments thereof with
high mannose, hybrid and complex type glycoforms at antibody-Fc
regions. The glyco-engineered antibodies may result in enhancement
of their effector functions, such as Fc.gamma.IIIA bindings and
antibody dependent cell mediated cytotoxicity (ADCC) etc., as well
as pharmacological properties. In addition, embodiments of the
present invention enable rapid investigation of effects of diverse
Fc glycosylations of therapeutic antibodies, particularly of those
highly sialylated complex type glycoforms that are expected to gain
anti-inflammatory activities, on their effector functions.
In one aspect, the present invention provides the EndoS2 mutants,
wherein the mutants have at least 80% homology thereto and exhibit
improved tranglycosylation activity on both fucosylated and
non-fucosylated GlcNAc acceptors against broad range of N-glycans
of high mannose, hybrid and complex types, wherein the said mutants
enable efficient transfer of an activated oligosaccharide donors on
fucosylated and non-fucosylated GlcNAc acceptors to form new
homogenous glycoform of glycopeptide or glycoprotein or therapeutic
antibodies.
In another aspect, the present invention provides EndoS2 mutants
that show remarkable transglycosylation activity but diminished
hydrolytic activity, wherein the mutants preferably includes site
specific mutations including mutations at T138, D182, D226, T227,
and T228, but are not limited to T138D (SEQ ID NO.6), T138E (SEQ ID
NO.7), T138F (SEQ ID NO.8), T138H (SEQ ID NO.9), T138K (SEQ ID
NO.10), T138L (SEQ ID NO.11), T138M (SEQ ID NO.12), T138N (SEQ ID
NO.13), T138Q (SEQ ID NO.14), T138R (SEQ ID NO.15), T138V (SEQ ID
NO.16), T138W (SEQ ID NO.17), D182Q (SEQ ID NO. 2), D226Q (SEQ ID
NO. 3), T227Q (SEQ ID NO. 4), and T228Q (SEQ ID NO. 5).
In a further aspects, the present invention provides for remarkable
transglycosylation activity of EndoS2 mutants but diminished
hydrolytic activity to transfer activated oligosaccharide donors to
fucosylated or non-fucosylated GlcNAc acceptors, wherein the
activated oligosaccharides donors comprising synthetic glycan
oxazolines. In one embodiment, the synthetic glycan oxazoline
comprising diverse N-glycans of high mannose, hybrid and complex
types having the formula:
##STR00001## Wherein, R.sup.1 is --H or N-acetyl glucosamine
attached via .beta.-1, 4 linkage and R.sup.2 and R.sup.3 are same
or different and are independently selected from the glycosyl
groups shown in FIG. 13.
In another aspect, the present invention provides EndoS2 mutants
for transglycosylation at core fucosylated or non-fucosylated
GlcNAc-acceptor, wherein the core fucosylated or non-fucosylated
GlcNAc-acceptor comprising core fucosylated or non-fucosylated
GlcNAc-peptides, proteins and IgG Fc domain or fragment
thereof.
In a further aspects, the present invention provides EndoS2 mutants
for transglycosylation at core fucosylated or non-fucosylated
GlcNAc-IgG, wherein the IgG is a monoclonal antibody and is
selected from the group consisting of cetuximab, rituximab,
muromonab-CD3, abciximab, daclizumab, basiliximab, palivizumab,
infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab,
ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, I-131
tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab,
natalizumab, etanercept, IGN101, volociximab, Anti-CD80 mAb,
Anti-CD23 mAb, CAT-3888, CDP-791, eraptuzumab, MDX-010, MDX-060,
MDX-070, matuzumab, CP-675,206, CAL, SGN-30, zanolimumab,
adecatumumab, oregovomab, nimotuzumab, ABT-874, denosumab, AM 108,
AMG 714, fontolizumab, daclizumab, golimumab, CNTO 1275,
ocrelizumab, HuMax-CD20, belimumab, epratuzumab, MLN1202,
visilizumab, tocilizumab, ocrerlizumab, certolizumab pegol,
eculizumab, pexelizumab, abciximab, and ranibizimumab.
In a separate aspect, the present invention provides a remodeling
method of core fucosylated or non-fucosylated GlcNAc-peptide,
protein, and IgG or IgG-Fc fragment, wherein the method comprising:
providing peptide/protein/antibody-GlcNAc acceptor or Fc fragment
and reacting with an activated oligosaccharide donors under the
catalysis of Streptococcus Pyogenes EndoS2 mutants, and thereby
preparing substantially pure glycoform of pre-existing peptides,
proteins and monoclonal antibodies having heterogeneous
glycosylation states.
In further aspect, the present invention provides method of using
EndoS2 mutants for glycan remodeling of therapeutic IgG or Fc
fragment thereof, wherein the method comprising: A. Treating
natural or recombinant core fucosylated or non-fucosylated
therapeutic IgG or IgG-Fc fragment carrying heterogeneous N-glycans
with Endoglycosidase (wild type EndoS2) together with or without
bacterial alpha fucosidases to hydrolyze bond between two reducing
end GlcNAc residues to form core fucosylated or non-fucosylated
GlcNAc-IgG acceptor; B. Transferring the wide range of predefined
oligosaccharide building units in the form of activated
oligosaccharide donors to core fucosylated or non-fucosylated
GlcNAc-IgG to reconstitute natural beta 1, 4 linkage through
transglycosylation using Streptococcus Pyogenes EndoS2 mutants,
thereby attaching the predefined oligosaccharide to remodel core
fucosylated or non-fucosylated IgG or Fc fragment thereof.
In further aspect, the present invention provides a composition of
fucosylated or non-fucosylated glyco-engineered antibodies or
antigen binding fragments comprising of IgG molecules having the
same N-glycan structure at each site of the Fc region, wherein the
N-glycan is of high mannose, hybrid, and complex types and is
selected from the group consisting of:
##STR00002## Wherein, R.sup.1 is --H or N-acetyl glucosamine
attached via .beta.-1, 4 linkage and R.sup.2 and R.sup.3 are same
or different and are independently selected from the glycosyl
groups shown in FIG. 13.
In another aspect, the present invention provide the
glycoengineered antibodies with improved effector functions such as
bindings to Fc.gamma.IIIA and ADCC, as compared to non-modified
antibodies.
Another aspect of the present disclosure features a pharmaceutical
composition comprising a composition of glyco-engineered antibodies
described herein and a pharmaceutically acceptable carrier for the
treatment of cancer in a patient.
Examples of cancers include, but not limited to, B cell lymphomas,
NHL, precursor B cell lymphoblastic leukemia/lymphoma and mature B
cell neoplasms, B cell chronic lymphocytic leukemia (CLL)/small
lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia,
lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular
lymphoma (FL), low-grade, intermediate-grade and high-grade (FL),
cutaneous follicle center lymphoma, marginal zone B cell lymphoma,
MALT type marginal zone B cell lymphoma, nodal marginal zone B cell
lymphoma, splenic type marginal zone B cell lymphoma, hairy cell
leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma,
plasmacytoma, plasma cell myeloma, post-transplant
lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and
anaplastic large-cell lymphoma (ALCL).
The present invention envisioned glycoengineering of antibodies
selected from the group consisting of cetuximab, rituximab,
muromonab-CD3, abciximab, daclizumab, basiliximab, palivizumab,
infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab,
ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, 1-131
tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab,
natalizumab, etanercept, IGN101, volociximab, Anti-CD80 mAb,
Anti-CD23 mAb, CAT-3888, CDP-791, eraptuzumab, MDX-010, MDX-060,
MDX-070, matuzumab, CP-675,206, CAL, SGN-30, zanolimumab,
adecatumumab, oregovomab, nimotuzumab, ABT-874, denosumab, AM 108,
AMG 714, fontolizumab, daclizumab, golimumab, CNTO 1275,
ocrelizumab, HuMax-CD20, belimumab, epratuzumab, MLN1202,
visilizumab, tocilizumab, ocrerlizumab, certolizumab pegol,
eculizumab, pexelizumab, abciximab, and ranibizimumab.
Other aspects and advantages of the invention will be apparent from
the following description and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows sequence of wild type EndoS2 and potential amino acid
residues selected for site directed mutagenesis (SEQ ID NO:1).
FIG. 2 shows amino acid sequence of EndoS2 mutants T138D (SEQ ID
NO.6), T138E (SEQ ID NO.7), T138F (SEQ ID NO.8), T138H (SEQ ID
NO.9), T138K (SEQ ID NO.10), T138L (SEQ ID NO.11), T138M (SEQ ID
NO.12), T138N (SEQ ID NO.13), T138Q (SEQ ID NO.14), T138R (SEQ ID
NO.15), T138V (SEQ ID NO.16), T138W (SEQ ID NO.17), D182Q (SEQ ID
NO. 2), D226Q (SEQ ID NO. 3), T227Q (SEQ ID NO. 4), and T228Q (SEQ
ID NO. 5) in accordance with one embodiment of the invention.
FIG. 3 shows (A) The carton represents the putative 3.sup.rd,
4.sup.th and 5.sup.th .beta.-sheets of the catalytic domain of Endo
S2 based on the alignment with EndoS. The catalytic residue E186,
colored in red, served as a general acid/base while the mutated
amino acids sites surrounding E186 are colored in blue. (B) The
glycan hydrolytic activity of wild type EndoS2 and selected mutants
at each site using commercial Rituximab as a substrate. The
reactions of EndoS2 mutants (52 nM) with commercial Rituximab (52
incubated for 120 min and analyzed by SDS-PAGE. The relative
percentage of original Rituximab (Rtx-glycan) is shown. Mutant
D184Q was used for comparison.
FIG. 4 shows the trans glycosylation activity of wild type EndoS2
and selected mutants (67.5 nM) at each sites using GlcNAc-Rituximab
(67.5 .mu.M) as an acceptor and .alpha.-2,6 sialylated bi-antennary
complex type glycan (SCT)-oxazoline (2.5 mM) as a donor. The
reaction was incubated for 2 hours and analyzed by SDS-PAGE. The
relative percentages of Rituximab with .alpha.-2,6 sialylated
bi-antennary complex type glycan at Fc region (Rtx-SCT) is shown.
Mutant D184Q was used for comparison.
FIG. 5 shows the transglycosylation activity comparison of mutants
at T138 using GlcNAc-Rituximab as an acceptor and .alpha.-2, 6
sialylated bi-antennary complex type glycan (SCT)-oxazoline as a
donor. The reactions containing T138 mutants (67.5 nM),
GlcNAc-Rituximab (67.5 .mu.M), and SCT-oxazoline (2.5 mM) were
incubated for 2 hours and analyzed by SDS-PAGE. The relative
percentages of Rituximab with .alpha.-2, 6 sialylated bi-antennary
complex type glycan at Fc region (Rtx-SCT) is shown.
FIG. 6 shows the transglycosylation activity of selected mutants of
EndoS2 against high mannose type Man.sub.5GlcNAc.sub.2 (glycan G1),
Man.sub.9GlcNAc.sub.2 (glycan G2) and hybrid series glycans (G3 and
G4), using GlcNAc-Rituximab as an acceptor and glycan-oxazolines
(G1-G4) as donor substrates. The reaction progress was monitored by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SD
S-PAGE). Lane 1: marker; Lane 2: GlcNAc-Rituximab; time points are
in Min.; product % is shown at the bottom.
FIG. 7 shows the transglycosylation activity of selected mutants of
EndoS2 against tri-antennary complex type glycans G5-with terminal
galactose residues and G6-with terminal .alpha.-2, 6 sialic acids.
The reaction was performed using GlcNAc-Rituximab as an acceptor
and glycan-oxazolines (G4-G5) as donor substrate. The reaction
progress was monitored by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). Lane 1: marker; Lane 2:
GlcNAc-Rituximab; time points are in Min.; product % is shown at
the bottom.
FIG. 8 shows the transglycosylation activity of selected mutants of
EndoS2 against series of bi-antennary complex type structures.
Glycan G7 is bi-antennary with terminal di-galactose, glycan G8 is
bi-antennary with bisected GlcNAc and terminal di-galactose, glycan
G9 is bi-antennary with alpha 1, 2 fucose on one of the two
terminal galactose, and glycan G10 is bi-antennary with alpha 1, 3
fucose on both GlcNAc and terminal di-galactose. The
transglycosylation was performed using GlcNAc-Rituximab as an
acceptor and glycan-oxazolines (G7-G10) as donor substrate. The
reaction progress was monitored by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). Lane 1: marker; Lane
2: GlcNAc-Rituximab; time points are in Min.
FIG. 9 shows transglycosylation activity of selected mutants of
EndoS2 against bi-antennary complex type glycans G7-with terminal
galactose residues and G16-with terminal .alpha.-2, 6 sialic acids.
The reaction was performed using Fuc(.alpha.-1, 6)-GlcNAc-Rituximab
as an acceptor and glycan-oxazolines (G4 and G16) as donor
substrate. The reaction progress was monitored by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SD S-PAGE). Lane 1:
marker; Lane 2: GlcNAc-Rituximab; time points are in Min.; product
% is shown at the bottom.
FIG. 10 shows the substrate specificity of selected mutants of
EndoS2 against broad range of high mannose, hybrid, and complex
type glycans.
FIG. 11 shows the structures of Rituximab with diverse glycoforms
at Fc region. The glycoforms includes series of high mannose,
hybrid, and bi- and tri-antennary complex type structures.
FIG. 12 shows SDS PAGE analysis of glycoengineered Rituximabs (Rtx
G1-G16), GlcNAc-Rituximab, and commercial Rituximab.
FIG. 13 shows structures of various glycosyl groups that can be
used with embodiments of the invention.
FIG. 14 shows structures of various glycosyl groups in glycan
oxazolines in accordance with embodiments of the invention.
FIG. 15 shows schematics of structures of glycoengineered
Rituximabs, (Rtx-G1-Rtx-G16) in accordance with embodiments of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the invention relate to selected mutants of EndoS2
that show remarkable transglycosylation activities to transfer a
broad range of N-glycans of high mannose, hybrid or complex types,
from activated oligosaccharide oxazolines to fucosylated or
non-fucosylated GlcNAc-peptides, proteins or IgGs with little or
negligible product hydrolysis. The novel EndoS2 mutants acted
efficiently to provide homogeneously glycosylated glycopeptides,
glycoproteins and therapeutic antibodies and Fc fragments thereof,
having various defined glycoforms. Still further, embodiments of
the present invention may provide glycoengineered antibodies with
enhancement of their effector functions, such as Fc.gamma.IIIA
bindings and antibody dependent cell mediated cytotoxicity (ADCC)
etc., as well as pharmacological properties. Embodiments of the
present invention also allow for rapid investigation of effects of
diverse Fc glycosylations of therapeutic antibodies on their
effector functions.
In the following description, reference is made to the accompanying
drawings that form a part hereof, and embodiments of the invention
are shown by way of specific examples which may be practiced. These
embodiments are described in detail to enable those skilled in the
art to practice the invention, and it is to be understood that
other embodiments may be devised and that structural, logical and
electrical changes may be made without departing from the scope of
the present invention. The following description of example
embodiments is, therefore, not to be taken in a limited sense, and
the scope of the present invention is defined by the appended
claims.
Unless defined otherwise, all technical and scientific terms used
herein have the same meanings as commonly understood by one of
ordinary skills in the art to which this invention belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, the preferred methods and materials are now
described. All publications and patents specifically mentioned
herein are incorporated by reference for all purposes including
describing and disclosing the chemicals, cell lines, vectors,
animals, instruments, statistical analysis and methodologies which
are reported in the publications which might be used in connection
with the invention. All references cited in this specification are
to be taken as indicative of the level of skill in the art. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
Before the present materials and methods are described, it is
understood that this invention is not limited to the particular
methodology, protocols, materials, and reagents described, as these
may vary. It is also to be under stood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims.
Definitions
It must be noted that as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural reference
unless the context clearly dictates otherwise.
As used herein, the term "glycan" refers to a polysaccharide,
oligosaccharide or monosaccharide. Glycans can be monomers or
polymers of sugar residues and can be linear or branched. A glycan
may include natural sugar residues (e.g., glucose,
N-acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose,
fucose, hexose, arabinose, ribose, xylose, etc.) and/or modified
sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6'
sulfo N-acetylglucosamine, etc).
As used herein, the terms "fucose," "core fucose," and "core fucose
residue" are used interchangeably and refer to a fucose in
.alpha.-1,6-position linked to the N-acetylglucosamine.
As used herein, the terms "N-glycan", "N-linked glycan", "N-linked
glycosylation", "Fc glycan" and "Fc glycosylation" are used
interchangeably and refer to an N-linked oligosaccharide attached
by an N-acetylglucosamine (GlcNAc) linked to the amide nitrogen of
an asparagine residue in a Fc-containing polypeptide. The term
"Fc-containing polypeptide" refers to a polypeptide, such as an
antibody, which comprises an Fc region.
As used herein, the term "glycosylation pattern" and "glycosylation
profile" are used interchangeably and refer to the characteristic
"fingerprint" of the N-glycan species that have been released from
a glycoprotein or antibody, either enzymatically or chemically, and
then analyzed for their carbohydrate structure, for example, using
LC-HPLC, or MALDI-TOF MS, and the like. See, for example, the
review in Current Analytical Chemistry, Vol. 1, No. 1 (2005), pp.
28-57; herein incorporated by reference in its entirety.
As used herein, the term "glycoengineered Fc" when used herein
refers to N-glycan on the Fc region has been altered or engineered
either enzymatically or chemically. The term "Fc glycoengineering"
as used herein refers to the enzymatic or chemical process used to
make the glycoengineered Fc.
The terms "homogeneous", "uniform", "uniformly" and "homogeneity"
in the context of a glycosylation profile of Fc region are used
interchangeably and are intended to mean a single glycosylation
pattern represented by one desired N-glycan species, with no trace
amount of precursor N-glycan
As used herein, the terms "IgG", "IgG molecule", "monoclonal
antibody", "immunoglobulin", and "immunoglobulin molecule" are used
interchangeably.
As used herein, the term "Fc receptor" or "FcR" describes a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
(see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).
FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92
(1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et
al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including
those to be identified in the future, are encompassed by the term
"FcR" herein. The term also includes the neonatal receptor, FcRn,
which is responsible for the transfer of maternal IgGs to the fetus
(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.
Immunol. 24:249 (1994)).
The term "effector function" as used herein refers to a biochemical
event that results from the interaction of an antibody Fc region
with an Fc receptor or ligand. Exemplary "effector functions"
include Clq binding; complement dependent cytotoxicity; Fc receptor
binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e.g. B
cell receptor; BCR), etc. Such effector functions can be assessed
using various assays known in the art.
As used herein, the term "Antibody-dependent cell-mediated
cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which
secreted Ig bound onto Fc receptors (FcRs) present on certain
cytotoxic cells (e.g. Natural Killer (NK) cells, neutrophils, and
macrophages) enable these cytotoxic effector cells to bind
specifically to an antigen-bearing target cell and subsequently
kill the target cell with cytotoxins. The antibodies "arm" the
cytotoxic cells and are absolutely required for such killing. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells is summarized
in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991). To assess ADCC activity of a molecule of interest,
an in vitro ADCC assay, such as that described in U.S. Pat. No.
5,500,362 or 5,821,337 may be performed. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998).
Approximately two-thirds of therapeutic proteins, available in the
market and/or currently in various stages of clinical trials are
monoclonal antibodies, of which there are 30 antibodies and their
derivatives have been approved for treatment of different
conditions mainly neoplastic diseases, inflammatory and auto
immunological diseases. However, the glycan microheterogeneity of
natural and recombinant glycoproteins presents a major barrier in
developing glycoprotein-based drugs. Controlling glycosylation
states is extremely difficult during protein or antibody expression
that are typically produced as a mixture of glycoforms that differ
only in the structure of the pendent oligosaccharides, which may
have different biological activities. Two enzymes that recently
have attracted great attention for glycoengineering of therapeutic
antibodies are EndoS and EndoS2 from the human pathogen
Streptococcus pyogenes (Collin and Olsen 2001; Sjogren et al.
2013). The enzymes were first discovered as bacterial immune
evasion factors that abolish the effector functions of
immunoglobulin G (IgG) by hydrolyzing N-linked glycans on the
antibody.
The complex N-linked oligosaccharide on each CH2 domain of IgGs is
crucial for the structure of the Fc region and thus the interaction
with the Fc receptors (Krapp et al. 2003; Woof and Burton 2004).
The oligosaccharide chain at IgG-Fc domain contains several
N-Acetyl-Glucosamine (GlcNAc) and mannose (Man) residues, and
eventually galactose (Gal) and fucose (Fuc) residues as well as
sialic acid (Sia or NANA for N-acetylneuraminic acid). A GlcNAc,
with or without al-6 Fuc, is attached to the Asn297. A
GlcNAc.beta.1-4 is attached to this first GlcNAc. A man.beta.1-4 is
then found, to which two Man.alpha.1-6 and Man.alpha.1-3 arms are
attached. Both arms contain an additional GlcNAc.beta.1-2 to which
a Gal.beta.1-4 can be attached or not. Thus, the carbohydrate chain
can contain 0, 1 or 2 galactose residues, defining G0, G1, and G2
glycoforms, respectively. Further variations occur, including the
presence of a bisecting GlcNAc.beta.1-4 and the capping of one or
both of the terminal galactose residues with a sialic acid or even
a Gal.alpha.1-3 residue. The enzymatic cleavage of the Fc-glycan
with Endoglycosidases causes the Fc region to deform, and thus,
dramatically decrease in IgGs binding to Fc.gamma. receptors
(Allhorn et al. 2008). Despite of their 37% sequence identity, both
EndoS and EndoS2 catalyze the hydrolysis of the .beta.-1,4 linkage
between the two N-acetylglucosamines (GlcNAcs) in the core of the
N-linked glycan of human IgG. However, in addition to complex type
glycans, EndoS2 hydrolyze hybrid and oligomannose structures to a
greater extent compared with EndoS (Sjogren et al. 2015).
Since the first antibody therapy was introduced in the 1980s, there
are more than 240 therapeutic antibodies in clinical trials and the
field is steadily expanding (Chan and Carter 2010). The role of the
IgG-Fc glycans on antibody functions has gained a huge attention in
the growing field of monoclonal therapeutic antibodies. Therefore,
to improve the efficacy of the therapeutic antibodies, the major
focus is turning towards the engineering the Fc-glycan that
specifically interact with selected Fc.gamma. receptors.
(Sondermann et al. 2013; Bournazos et al. 2014; Monnet et al. 2014;
Quast and Lunemann 2014). Some of the important glycan
modifications that dramatically affect the effector functions
includes, i) the lack of a core fucose residue attached to the
reducing end GlcNAc residue leads to increased affinity for
Fc.gamma.RIIIa and thus increased antibody-dependent cytotoxicity
(Iida et al. 2006); ii) sialic acid rich glycans on IgG that have
been claimed to increase the anti-inflammatory response of IgGs
through increased interaction with DC-SIGN receptors on dendritic
cells and macrophages (Anthony et al. 2008; Anthony and Ravetch
2010; Pincetic et al. 2014); iii) having bisecting GlcNAc induces a
strong ADCC as compared to its parental counterpart. The recent
improvements in biotechnology tools to control the Fc-glycosylation
states of IgG facilitates development of therapeutic antibodies
with predefined glycoforms. Accordingly, the EndoS2 mutants of
present invention is a great advancement in the field for
glyco-engineering of peptides, proteins, and antibodies of interest
to attach broad range of N-glycans of high mannose, hybrid and
complex types for functional and structural studies.
The features and advantages of the present invention are more fully
shown by the following non-limiting examples. One skilled in the
art would appreciate that these examples are for illustration only
and that other modifications and variations are possible without
departing from the scope of the invention.
Examples
Generation of EndoS2 Mutants for Glycoengineering of Peptides,
Therapeutic Proteins and Intact IgGs or Fc Fragment Thereof
Until now, examples of glycosynthases have been produced from some
GH85 endoglycosidases (ENGases), including EndoA, EndoM, and EndoD,
by site-directed mutagenesis of a key asparagine (Asn) residue
responsible for promoting oxazolinium ion intermediate formation
during hydrolysis.
EndoS from Streptococcus pyogenes belongs to the glycoside
hydrolase family 18 (GH18), which also includes EndoF1, EndoF2, and
EndoF3. These GH18 enzymes are known for their efficient hydrolytic
activities that cleave asparagine-linked bi-antennary glycans on
human IgGs to produce mono-GlcNAc antibodies. Even though EndoS can
also function as glycosynthases to synthesize chitobiose linkages
using glycan oxazolines as substrates, the intrinsic hydrolysis
activities of these enzymes present a major hurdle, which leads to
significantly reduced yields of synthetic glycoproteins. Further
improvement in enzymatic activities leads to the development of
endoglycosidase mutants, including EndoS D233Q. This gives
important motifs on the active site of ENGase: D-X-E in GH18. This
motif supports the catalysis mechanism on ENGase, which uses a
double-displacement reaction with neighboring group participation.
In this mechanism, the 2-acetamide group of the GlcNAc acts as a
nucleophile to substitute the leaving group at the anomeric center,
with the formation of an oxazolinium ion intermediate promoted by
the carboxylate of Asp. The catalytic residue, glutamate, acts as a
general acid/base that protonates the leaving aglycan group and
deprotonates the nucleophilic H.sub.2O causing the hydrolysis of
the oxazolinium ion intermediate to form the hydrolytic product.
The carboxylate of aspartate, 2 residues on the N-terminal side of
E, was proposed to promote and stabilize the formation of the
oxazolinium ion intermediate. The mutation on this D of EndoS to Q
was demonstrated to improve transglycosylation and diminish
hydrolysis activity.
Although the mutants EndoS D233Q and D233A demonstrated a great
potential for the synthesis of homogeneous antibody, the addition
of large amount of enzymes is required to achieve efficient
reaction. The preparation of enzymes and the following steps to
remove enzymes after reaction became tedious and labor intensive.
Recently, a new enzyme, EndoS2, was identified from another
serotype of Streptococcus pyogenes. In addition to the
endo-.beta.-N-acetylglucosaminidase activity on complex type of
N-glycan as in EndoS-catalyzed possess, EndoS2 can cleave hybrid
and oligomannose structures to a greater extent than EndoS
(Jonathan et al., 2013 and 2015).
EndoS2 shares only 37% sequence identity with EndoS, the structure
of which adopts a common ((.beta./.alpha.).sub.8 barrel
conformation in the catalytic domain. Based on alignment of these
two enzymes, residue E186 located on the fourth .beta.-sheet of
EndoS2 corresponds to the general acid/base D235 of EndoS.
In order to explore the catalytic efficiency of transglycosylation
of EndoS2, we set out to test whether conversion of amino acids
near catalytic site would modulate the trasglycosylation activity.
A few residues in the proximity of catalytic domain were chosen and
mutated by site-directed mutagenesis. The mutated residues include
T138 on the third .beta.-sheet, D182 on the fourth .beta.-sheet,
and D226, T227 and T228 on the fifth .beta.-sheet (FIG. 3A). The
EndoS2 mutants tested include T138D (SEQ ID NO.6), T138E (SEQ ID
NO.7), T138F (SEQ ID NO.8), T138H (SEQ ID NO.9), T138K (SEQ ID
NO.10), T138L (SEQ ID NO.11), T138M (SEQ ID NO.12), T138N (SEQ ID
NO.13), T138Q (SEQ ID NO.14), T138R (SEQ ID NO.15), T138V (SEQ ID
NO.16), T138W (SEQ ID NO.17), D182Q (SEQ ID NO. 2), D226Q (SEQ ID
NO. 3), T227Q (SEQ ID NO. 4), and T228Q (SEQ ID NO. 5) (FIG. 2 and
Table 1). These mutants expressed in E. coli in high yield as
His-tag fusion proteins, which can be purified with a Ni-NTA
affinity column. It has been observed that mutations near active
sites dramatically improved the transglycosylation activity and
decreased the endoglycosidase activity, resulting in remarkable
glycosynthase efficiency. In addition, these mutants are capable of
transferring complex type N-glycans from activated glycan
oxazolines to deglycosylated intact antibodies with negligible
product hydrolysis.
TABLE-US-00001 TABLE 1 below shows the sequences of various EndoS2
mutants. SEQ ID NO. 1 (Wild-type) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVFHDHTASDSPFWSELKDSYVHKLHQQGTALVQTIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVFKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQFMIGFSF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDFVFGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIFDGEKSDRFFTWGQTNWI
AFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 6 (T138D) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPFWSELKDSYVHKLHQQGTALVQDIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDRFFTWGQTNWI
AFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 7 (T138E) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPEWSELKDSYVHKLHQQGTALVQEIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESE
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDREFTWGQTNWI
AFDLGEINLAKEWRLENAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 8 (T138F) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILEVEHDHTASDSPFWSELKDSYVHKLHQQGTALVQFIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESE
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDREFTWGQTNWI
AFDLGEINLAKEWRLENAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIENSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 9 (T138H) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPEWSELKDSYVHKLHQQGTALVQHIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESE
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDREFTWGQTNWI
AFDLGEINLAKEWRLENAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 10 (T138K) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPEWSELKDSYVHKLHQQGTALVQKIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDFVFGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDRFFTWGQTNWI
AFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 11 (T138L) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPFWSELKDSYVHKLHQQGTALVQLIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDREFTWGQTNWI
AFDLGEINLAKEWRLENAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIENSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 12 (T138M) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPFWSELKDSYVHKLHQQGTALVQMIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDREFTWGQTNWI
AFDLGEINLAKEWRLENAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 13 (T138N) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILEVEHDHTASDSPEWSELKDSYVHKLHQQGTALVQNIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESE
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDRFFTWGQTNWI
AFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIENSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 14 (T138Q) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPEWSELKDSYVHKLHQQGTALVQQIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDRFFTWGQTNWI
AFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 15 (T138R) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPEWSELKDSYVHKLHQQGTALVQRIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDFVFGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDRFFTWGQTNWI
AFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 16 (T138V) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPEWSELKDSYVHKLHQQGTALVQVIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDREFTWGQTNWI
AFDLGEINLAKEWRLENAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIENSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID NO. 17 (T138W) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPEWSELKDSYVHKLHQQGTALVQWIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDREFTWGQTNWI
AFDLGEINLAKEWRLENAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIENSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID. 2 (D182Q) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPEWSELKDSYVHKLHQQGTALVQTIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLQIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIFDGEKSDRFFTWGQTNWI
AFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID. 3 (D226Q) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVFHDHTASDSPEWSELKDSYVHKLHQQGTALVQTIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMQTTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQFMIGFSF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDFVFGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIFDGEKSDRFFTWGQTNWI
AFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID. 4 (T227Q) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPEWSELKDSYVHKLHQQGTALVQTIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVEKEIAQLIGKNGSDKSKLLIMDQTLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQEMIGESF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDRFFTWGQTNWI
AFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843 SEQ ID. 5 (T228Q) 1
MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKT
DQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMA
EVPKEVDILFVEHDHTASDSPFWSELKDSYVHKLHQQGTALVQTIGVN
ELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFT
NKRTPEEDARALNVFKEIAQLIGKNGSDKSKLLIMDTQLSVENNPIFK
GIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQFMIGFSF
FEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKA
GIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKT
LMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGD
KIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGM
TGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKL
LMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEH
DILTDEVEGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFR
KDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLN
IGSGAIMMENLAKGAKVIGTSGDFEQAKKIEDGEKSDREFTWGQTNWI
AFDLGEINLAKEWRLENAETNTEIKTDSSLNVAKGRLQILKDTTIDLE
KMDIKNRKEYLSNDENWTDVAQMDDAKAIENSKLSNVLSRYWRFCVDG
GASSYYPQYTELQILGQRLSNDVANTLD 843
In accordance with embodiments of the invention, a novel EndoS2
mutant comprises a sequence selected from the sequences of SEQ ID
NOs. 2-17. These mutants show improved tranglycosylation activities
and reduced hydrolyzing activities. Therefore, they can catalyze
efficient transfer of activated oligosaccharide donors to core
GlcNAc-acceptors, which may be fucosylated or non-fucosylated.
In accordance with some preferred embodiments, an EndoS2 mutant may
have a sequence identity of at least 80% (e.g., 80%, 85%, 90%, 95%,
or 98%) to a sequence in SEQ ID Nos. 2-17 and have the desired
transglycosylation activity, or fragment thereof having the
transglycosylation activity.
In other preferred embodiments, the EndoS2 mutants of this
invention, wherein the mutation sites are located in a region
selected from the group consisting of residues 133-143, residues
177-187, and residues 221-233.
In yet other preferred embodiments, the EndoS2 mutants of this
invention include T138D (SEQ ID NO.6), T138E (SEQ ID NO.7), T138F
(SEQ ID NO.8), T138H (SEQ ID NO.9), T138K (SEQ ID NO.10), T138L
(SEQ ID NO.11), T138M (SEQ ID NO.12), T138N (SEQ ID NO.13), T138Q
(SEQ ID NO.14), T138R (SEQ ID NO.15), T138V (SEQ ID NO.16), T138W
(SEQ ID NO.17), D182Q (SEQ ID NO. 2), D226Q (SEQ ID NO. 3), T227Q
(SEQ ID NO. 4), and T228Q (SEQ ID NO. 5).
The Glycan Hydrolytic Activity of EndoS2 and its Mutants
The glycan hydrolytic activities of EndoS2 mutants were measured by
using commercial Rituximab as a substrate. Rituximab, a therapeutic
anti-CD20 monoclonal antibody, was used as a model mAb to examine
the hydrolytic activity and potential transglycosylation activity
of the EndoS2 mutants. The major Fc glycans of commercial Rituximab
are core-fucosylated biantennary complex type oligosaccharides
carrying 0-2 galactose moieties named G0F, G1F, and G2F glycoforms.
Rituximab was treated with the wild type EndoS2 and EndoS2 mutants
in a molar ratio of 1:1000 (enzyme: Rituximab). The glycan
hydrolysis process was monitored by sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE).
The treatment with wild type EndoS2 resulted in a rapid
deglycosylation to produce the corresponding GlcNAc-Fc N-glycans at
the glycosylation sites (N297). These results confirm the
remarkable Fc glycan-hydrolyzing activity of the wild-type EndoS2
on intact IgG, implicating its usefulness in the first step
(hydrolysis) for glycosylation remodeling of mAbs. However,
treatment with EndoS2 mutants showed reduced hydrolytic activities,
as compared to the wild-type (WT) EndoS2. In particular, mutants at
T138 and D226 exhibited extremely low or almost no N-glycan
hydrolysis abilities during a 2 hour incubation period, and
mutation at D182Q showed reduced the reaction rate by more than 60
folds, as compared to the WT EndoS2. These results indicate that
residues D182, T138 and D226 are critical for the glycoside
hydrolase activity (FIG. 3).
Transglycosylation Potentials of EndoS2 and its Mutants Using 2,6
Sialylated Bi-Antennary Complex Type (SCT) Oxazolines as the Donor
Substrates
Transglycosylation abilities of EndoS2 and its mutants were then
examined using the GlcNAc-Rituximab as an acceptor and
alpha-2,6-sialylated bi-antennary complex type (SCT) oxazolines as
a donor substrate, as depicted in FIG. 4. The glycosylation
remodeling process was monitored by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). Incubation of the
GlcNAc-Rituximab and the SCT-oxazoline (donor/acceptor, 1:1, weight
ratio) with the wild type EndoS2 and its mutants (enzyme/antibody,
1:1000, molar ratio) showed the transient formation of the
corresponding transglycosylation products for the wild type EndoS2
as monitored by SDS-PAGE, probably due to quick in situ hydrolysis
of the products by the wild-type enzyme. Despite the loss of
hydrolysis ability, all selected mutants (T138Q, D182Q, D226Q,
T227Q and T228Q) had significant transglycosylation activity. These
results indicate that the EndoS2 mutants are new efficient
glycosynthases that enable the glycosylation of deglycosylated
intact IgG with complex type N-glycan without product
hydrolysis.
Effects of Various Amino Acid Residues at T138 on their
Transglycosylation Activities
To identify the optimum amino acid residue at the site T138 that
show potent transglycosylation activity only but devoid of
hydrolytic activity, various mutations at this site were performed.
The transglycosylation abilities of these mutants were then
examined using the GlcNAc-Rituximab as an acceptor and
alpha-2,6-sialylated bi-antennary complex type (SCT) oxazolines as
a donor substrate (FIG. 5). Various mutants at T138 (67.5 nM) were
incubated with 67.5 .mu.M GlcNAc-Rituximab and 2.5 mM SCT-oxazoline
for 2 hr. Products were analyzed by SDS-PAGE, and the relative
percentages of GlcNAc-Rituximab (IgG) and Rituximab-SCT
(IgG-glycan) are shown in FIG. 5. Among the mutants tested, T138E,
T138R, T138W, T138M, and T138Q provided maximum tranglycosylation
potency.
Transglycosylation Efficiencies of EndoS2 Mutants Using a Broad
Range of N-Glycans of High Mannose, Hybrid and Complex Types
Human IgGs molecules contain N-glycan on each of their Fc CH2
domains. These glycans include high-mannose, hybrid, and complex
types. It has been demonstrated that the compositions Fc N-glycans
are important determinants of the pro- and anti-inflammatory
activities of antibodies. For example, the lack of the core fucose,
as well as the attachment of a bisecting GlcNAc moiety,
dramatically enhances the affinity of antibody for the
Fc.gamma.IIIa receptor (Fc.gamma.RIIIa), which is responsible for
the antibody-dependent cellular cytotoxicity (ADCC). The
recombinant IgG molecules containing high-mannose glycans have been
shown to clear faster in human blood, and exhibit decreased thermal
stability. In addition, IgG molecules containing high-mannose and
hybrid glycans showed more conformational flexibility in the CH2
domain. The most routine way to produce IgGs with distinct high
mannose, hybrid and complex type Fc-glycans is to use various
expression systems, including mammalian, plant, and yeast host
cells. However, such expression systems often provide a mixture of
glycoforms rather than a single glycan structure. Therefore,
obtaining the pure glycoforms of therapeutic antibodies for
biophysical and structural studies is of great interest in the
glycan engineering field. Accordingly, glycosynthases, which enable
efficient transfer of distinct high mannose, hybrid and complex
type glycan to GlcNAc acceptors of peptide, proteins, and IgGs, are
needed.
Next, the transglycosylation abilities of EndoS2 mutants of this
invention were accessed using series of high mannose hybrid and
complex types glycan oxazolines (Table 2). Results suggest that, in
addition to the sialylated complex type N-glycan oxazolines, the
EndoS2 mutants were equally efficient at transferring high mannose
series Man.sub.5GlcNAc-oxazoline (G1), Man.sub.9GlcNAc-oxazoline
(G2), hybrid series glycan G3 and G4, and bi- and tri-antennary
complex series glycans G5-G16 for antibody glycoengineering,
leading to the formation of the corresponding homogeneous
glycoforms. Rituximab was used as a model antibody for this study.
However, one skilled in the art would appreciate that other
glycoproteins can also be modified in a similar manner. These
results suggest that, in addition to the sialylated complex type
N-glycan oxazolines, the EndoS2 mutants were equally efficient at
transferring high mannose, hybrid, and triantennary complex type
glycans.
Table 2 Lists the Structures of Diverse Glycan Oxazolines Used to
Assess Transglycosylation Activity of Selected Mutants of
EndoS2
TABLE-US-00002 TABLE 2 ##STR00003## Glycan oxazoline general
formula Glycan Type Glycan number High mannose type G1 G2 Hybrid
Type G3 G4 Tri-antennary Complex G5 Type G6 Bi-antennary Complex G7
Type G8 G9 G10 G11 G12 G13 G14 G15 G16 Note: the structures of
G1-G16 are shown in FIG. 14.
The complex type glycans are divided into bi-, tri-, and
tetra-antennary types based on the number of antennae present on
the Man-GlcNAc-GlcNAc core. It is quite unusual that a
glyocosynthase that accepts bi-antennary glycan-oxazoline can also
accept tri- or tetra-antennary glycan-oxazolines because of the
bulky nature of these glycans. In order to prove the effectiveness
of these novel EndoS2 mutants, a series of complex type glycan
oxazolines (Table 2) were used for transglycosylations, wherein
Rituximab was used as a model antibody. The results are shown in
FIGS. 7-8. These results indicate the remarkable transglycosylation
efficiencies of the EndoS2 glycosynthase mutants to transfer
diverse high mannose, hybrid and complex type glycans to an IgG-Fc
region. Therefore, the EndoS2 glycosynthase mutants of the
invention can be used for efficient and complete transglycosylation
of different therapeutic antibodies for enhancement of their
effector functions, because these novel mutants are devoid of (or
with minimal) product hydrolytic activities.
The present invention discloses selected mutants of EndoS2 that
show excellent transglycosylation activities with a broad range of
N-glycans, including high mannose, hybrid and complex types.
In preferred embodiments, N-glycans of high mannose, hybrid and
complex types are in an active oxazoline form, as shown by the
general formula in Table 2.
In some embodiments, the high mannose type N-glycans described
herein are selected from group consisting of Man.sub.3GlcNAc,
Man.sub.5GlcNAc, Man.sub.6GlcNAc, Man.sub.7GlcNAc, Man.sub.8GlcNAc,
and Man.sub.9GlcNAc. In preferred embodiments, the high mannose
type N-glycan is Man.sub.5GlcNAc.
In some embodiments, the hybrid type N-glycans described herein
comprise at least one .alpha.-2,6- or .alpha.-2,3 terminal sialic
acid on the alpha-1,3 arm, wherein the alpha-1,6 arm contains the
trimannose residues.
In some embodiments, the hybrid type N-glycans described herein
comprise at least one terminal galactose on the alpha-1,3 arm,
wherein the alpha-1,6 arm contains the trimannose residues.
In some embodiments, the hybrid type N-glycans described herein
comprise at least one terminal GlcNAc on the alpha-1,3 arm, wherein
the alpha-1,6 arm contains the trimannose residues.
In some embodiments, the complex type glycans are of bi-, tri- and
tetra-antennary complex types.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprise at least one .alpha.-2,6 or .alpha.-2,3
terminal sialic acid. In preferred embodiments, the N-glycans
comprise two .alpha.-2,6 and/or .alpha.-2,3 terminal sialic
acids.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprise at least one terminal galactose or
GlcNAc. In preferred embodiments, the N-glycans comprise two
terminal galactose and/or GlcNAc.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprise at least one alpha-1,2-fucose. In
preferred embodiments, the N-glycans comprise two
alpha-1,2-fucoses.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprise at least one alpha-1,3-fucose. In
preferred embodiments, the N-glycans comprise two
alpha-1,3-fucose.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprise bisecting GlcNAc.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprise at least one LacNAc repeat unit. In
preferred embodiments, the N-glycans comprise two LacNAc repeat
units.
In some embodiments, the tri-antennary complex type N-glycans
described herein comprise at least one .alpha.-2,6 or .alpha.-2,3
terminal sialic acid. In preferred embodiments, the N-glycans
comprise three .alpha.-2-6 and/or .alpha.-2,3 terminal sialic
acids.
In some embodiments, the tri-antennary complex type N-glycans
described herein comprise at least one terminal galactose or
GlcNAc. In preferred embodiments, the N-glycans comprise three
terminal galactose and/or GlcNAc.
In some embodiments, the complex type glycans are of bi-, and
triantennary complex types comprising asymmetric antennae on either
the alpha-1,3 or alpha-1,6 arm.
In some embodiments, the hybrid and bi-, and triantennary complex
type N-glycans described herein comprise .alpha.-2,6 or .alpha.-2,3
terminal sialic acid. In a preferred embodiments, the hybrid and
bi-, and triantennary complex type N-glycan comprises .alpha.-2,6
terminal sialic acid.
Glycoengineering of Rituximab to Provide Diverse Non-Fucosylated
Glycoform for Improvement of Effector Functions
Rituximab is a monoclonal antibody targeting the CD20 protein which
is primarily found on the surface of 95% of B cell lymphomas.
Rituximab destroys B cells and is therefore used to treat diseases
which are characterized by excessive numbers of B cells, overactive
B cells, or dysfunctional B cells. Rituximab is produced in Chinese
hamster ovary (CHO) cells often delivers heterogeneous mixtures of
glycosylation patterns, which may not show similar biological
properties. Diversity in Fc glycosylation within an antibody may
lead to diversity in Fc effector functions. Thus, this
heterogeneity in Fc glycans has a functional consequence as it
influences binding of IgG molecules to Fc receptors and Clq and
thereby impacts antibody effector functions, and may trigger
undesired effects in patients, which would be a safety concern.
Therefore, there is a need for improving monoclonal antibody
therapy with improved anti-CD20 antibodies. A few specific
glycoforms in the heterogeneous mixtures of glycosylation patterns
are known to confer desired biological functions. Furthermore, in
case of complex type glycoforms, apart from few modifications such
core fucose and bisecting GlcNAc, several native modifications such
as alpha-1,2 fucose on outer GlcNAc, alpha-1,3 fucose on Galactose,
poly LacNAc motifs, and tri- and tetraantennary, have never been
explored for their effects on biological activities. Thus, it is of
great interest to generate therapeutic antibodies containing a
well-defined glycan structure and sequence as desired glycoforms
for therapeutic purposes.
Described herein are the functionally active anti-CD20
glycoengineered antibodies with optimized glycoforms that exhibit
more potent biological activities, as compared to the therapeutic
monoclonal antibodies.
The present disclosure features a novel class of anti-CD20
antibodies that can be generated from anti-CD20 monoclonal
antibodies by Fc glycoengineering. The individual anti-CD20
glycoenginnered antibodies comprise homogeneous population and
contain the same Fc glycan with a well-defined glycan structure and
sequence. The glycoengineered anti-CD20 antibodies according to the
present invention specifically bind to the same epitope of a human
CD20 antigen on a cell membrane as its parental antibody. In
addition, the homogeneous population of the same antibody all have
the same effector binding site.
The term "parental antibody" as used herein refers to the anti-CD20
monoclonal antibody used to produce an anti-CD20 glycoengineered
antibody. The parental antibodies can be obtained by cell culturing
such as mammalian cell culture, Pichia pastoris or insect cell
lines. Preferably, the parental antibodies are produced in
mammalian cell culture. Exemplary parental antibodies include, but
not limited to, Rituximab, Ofatumumab, Tositumomab, Ocrelizumab,
11B8 or 7D8 (disclosed in WO2004/035607).
In some embodiments, the exemplary anti-CD20 glycoengineered
antibodies described herein comprise a heavy chain having the amino
acid sequence set forth in SEQ ID NO.: 18, and a light chain having
the amino acid sequence set forth in SEQ ID NO: 19. In preferred
embodiments, the anti-CD20 glycoengineered antibodies each comprise
a light chain sequence and a heavy chain sequence of Rituximab.
Table 3 below shows the heavy chain and the light chain sequences
of Rituximab.
TABLE-US-00003 TABLE 3 Rituximab Accession Number: DB00073 Source:
http://www.drugbank.ca/drugs/DB00073 Rituximab heavy chain SEQ ID
NO: 18: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWI
GAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC
ARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV
PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK SEQ ID NO: 19 Rituximab light chain
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIY
ATNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTF
GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC
In some embodiments, the N-glycan is attached to the Asn-297 of the
Fc region of Rituximab.
The N-glycans according to the invention may have high mannose,
hybrid and bi- and tri-antennary complex type structures, wherein
the N-glycan structures having general formula:
##STR00004##
wherein, R.sup.1 is --H or N-acetyl glucosamine attached via a
.beta.-1,4 linkage and R.sup.2 and R.sup.3 are same or different
and are independently selected from the glycosyl groups shown in
FIG. 13.
In some embodiments, the N-glycans described herein may have
additional intrachain substitutions comprising "bisecting" GlcNAc,
.alpha.-1,2 fucose, .alpha.-1,3 fucose, with or without an
.alpha.-2,3 and/or .alpha.-2,6 sialic acids, and may be extended
poly LacNAc motifs.
In some embodiments, the high mannose type N-glycan described
herein are selected from group consisting of Man.sub.3GlcNAc,
Man.sub.5GlcNAc, Man.sub.6GlcNAc, Man.sub.7GlcNAc, Man.sub.8GlcNAc,
and Man.sub.9GlcNAc. In preferred embodiments, the high mannose
type N-glycan is Man.sub.5GlcNAc.
In some embodiments, the hybrid type N-glycans described herein
comprise at least one .alpha.-2,6 or .alpha.-2,3 terminal sialic
acid, or at least one terminal galactose, or at least one terminal
GlcNAc on the alpha-1,3 arm, while the alpha-1,6 arm contains
trimannose residues.
In some embodiments, the complex type glycans are of bi-, tri- or
tetra-antennary complex types.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprise at least one .alpha.-2,6 or .alpha.-2,3
terminal sialic acid. In preferred embodiments, the N-glycans
comprise two .alpha.-2,6 and/or .alpha.-2,3 terminal sialic
acids.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprise at least one terminal galactose or
GlcNAc. In preferred embodiments, the N-glycans comprise two
terminal galactose and/or GlcNAc.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprise at least one alpha-1,2 fucose. In
preferred embodiments, the N-glycans comprise two alpha-1,2
fucoses.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprises at least one alpha-1,3 fucose. In
preferred embodiments, the N-glycans comprise two alpha-1,3
fucoses.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprise bisecting GlcNAc.
In some embodiments, the bi-antennary complex type N-glycans
described herein comprise at least one LacNAc repeat unit. In
preferred embodiments, the N-glycans comprise two LacNAc repeat
units.
In some embodiments, the tri-antennary complex type N-glycans
described herein comprise at least one .alpha.-2,6 or .alpha.-2,3
terminal sialic acid. In preferred embodiments, the N-glycans
comprise three .alpha.-2,6 and/or .alpha.-2,3 terminal sialic
acids.
In some embodiments, the tri-antennary complex type N-glycans
described herein comprise at least one terminal galactose or
GlcNAc. In preferred embodiments, the N-glycans comprise three
terminal galactose and/or GlcNAc.
In some embodiments, the complex type glycans are of bi-, tri- or
tetra-antennary complex types, comprising asymmetric antennae on
either the alpha-1,3 or alpha-1,6 arm.
In some embodiments, the hybrid and bi- or tri-antennary complex
type N-glycans described herein comprise .alpha.-2,6 or .alpha.-2,3
terminal sialic acid. In a preferred embodiments, the hybrid and
bi-, tri- and tetra-complex type N-glycans comprise .alpha.-2,6
terminal sialic acid.
Preferrably, the N-glycans according to embodiments of the
invention are free of core fucose.
Table 4 lists exemplary N-glycans in anti-CD20 glycoengineered
antibodies. Embodiments of the present disclosure may include or
exclude any of the N-glycans listed herein.
TABLE-US-00004 TABLE 4 Glycan Type Glycoengineered Rituximab High
mannose type Rtx-G1 Rtx-G2 Hybrid Type Rtx-G3 Rtx-G4 Tri-antennary
Complex Type Rtx-G5 Rtx-G6 Bi-antennary Complex Type Rtx-G7 Rtx-G8
Rtx-G9 Rtx-G10 Rtx-G11 Rtx-G12 Rtx-G13 Rtx-G14 Rtx-G15 Rtx-G16
Note: the structures of Rtx-G1-Rtx-G16 are shown in FIG. 15.
Biological Functions of Glycoengineered Rituximab
Fc-glycosylation of a variety of antibodies greatly influence the
effector-mediated functions, including ADCC, CDC, and circulating
half-life. ADCC enhancement is a key strategy for improving
therapeutic antibody drug efficacy. It has the potential of
lowering effective drug dosage for the benefits of lower drug cost.
The glycoengineered anti-CD20 antibodies described herein have cell
growth inhibitory activities including apoptosis against human CD20
expressing cells. In some embodiments, the glycoengineered
anti-CD20 antibodies exhibits more potent cell growth inhibitory
activities, as compared to its parent antibody.
Binding Between Fc.gamma.RIIIA and Glycoengineered Rituximab
Table 5 lists exemplary Fc.gamma.RIIIA binding of anti-CD20
glycoengineered antibodies and Rituximab.
TABLE-US-00005 TABLE 5 Bindings of Fc.gamma.RIIIA to
glycoengineered Rituximabs using ELISA EC.sub.50 EC.sub.50 Maxi
binding improvement Glycoform (ng/mL) (A450 nm) Folds Note
Commercial 1045 1.2 1 Category Rituximab A: Rtx-G1 160 1.4 6.5
increase Rtx-G2 1050 1.1 ~1 >30% Rtx-G3 320 1.3 3.2 Category
Rtx-G4 394 1.2 2.7 B: Rtx-G5 31 2.6 35 increase Rtx-G6 32 2.8 34
>15-30% Rtx-G7 31 2.5 35 Category Rtx-G8 34 2.3 32 C: Rtx-G9 61
2.4 17 increase Rtx-G10 43 2.2 26 >5-10% Rtx-G11 199 2.4 5.4
Category Rtx-G12 33 2.6 33 D: No Rtx-G13 48 2.6 22 change Rtx-G14
33 2.4 33 Rtx-G15 34 2.3 32 Rtx-G16 32 2.5 34
Fc.gamma.RIIIA binding may be measured using assays known in the
art. Exemplary assays are described in the examples. The Fc
receptor binding may be determined as the relative ratio of
anti-CD20 glycoengineered antibodies vs. Rituximab. Fc receptor
binding in exemplary embodiments is increased by at least 2.5-fold,
3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,
11-fold or 20-fold, 30-fold or higher. High mannose series
Man.sub.9GlcNAc2 glycoforms are unable to offer promising binding.
However, Man.sub.5GlcNAc2 glycoform was better than the
non-modified Rituximab. Both hybrid type glycoforms could only
achieve 2-3 folds enhanced binding to Fc.gamma.RIIIA Interestingly,
all the complex type glycoforms, including bi- and tri-antennary
types, provided more than 30-folds enhanced bindings to
Fc.gamma.RIIIA, as compared to the original Rituximab. Having
alpha-1,3 fucose on GlcNAc showed no major effect on the
Fc.gamma.RIIIA binding. However, 1,2 fucose slightly reduced the
binding. Furthermore, having multiple antennae, such as those
present in the tri-antennary glycoform, showed no major
contribution in the binding. However, it is believed that glycans
with increased sialic acid contents might gain anti-inflammatory
activities.
As compared to Rituximab, the binding data showed that the
glycoengineered anti-CD20 antibodies, particularly those having
complex glycoforms, exhibit stronger binding affinities for the
Fc.gamma.RIIIA
ADCC Activities of Glycoengineered Rituximab
The ADCC activity of the glycoengineered rituximab's according to
the invention is at least 3 fold increased, preferably at least 9
fold, more preferably at least 10 fold increased ADCC activity,
preferably at least 12 fold increased ADCC activity, preferably at
least 20 fold increased ADCC activity, most preferred at least 30
fold increased ADCC activity compared to the ADCC activity of the
parental antibody.
Table 6 lists exemplary enhanced ADCC activities of selected
glycoengineered anti-CD20 antibodies and commercial Rituximab.
Exemplary assays are described in the examples.
TABLE-US-00006 TABLE 6 EC.sub.50 Maximal cell Glycoform R.sup.2
[ng/mL] lysis (%) Folds Commercial 0.987 13.55 27.79 1 Rituximab
Rtx-G5 0.946 2.181 32.02 6.55 Rtx-G6 0.954 2.927 39.18 4.6 Rtx-G14
0.942 2.323 38.76 5.8 Rtx-G15 0.968 1.911 37.98 7.0
A series of anti-CD20 glycoengineered antibodies disclosed by
present invention, in particular those with complex glycoforms,
exhibit enhanced ADCC activities, as compared to the parental
antibody, Rituximab. It is contemplated that the glycoengineered
antibodies of the invention may exhibit superior effect as
therapeutic agents for B cell-mediated malignant tumors and
immunological diseases, in which B cells or antibodies produced by
B cells are involved. An object of the present invention is to use
the anti-CD20 glycoengineered antibodies in development of
therapeutic agents.
Taken together, anti-CD20 glycoengineered antibodies, exhibit
enhanced ADCC activities and stronger Fc.gamma.RIIIA binding
affinities, as compared to Rituximab. The glycoantibodies of the
invention may provide a superior clinical response either alone or,
in a composition comprising two or more such antibodies, and
optionally in combination with other treatments such as
chemotherapy. The ADCC-enhanced anti-CD20 glycoengineered antibody
may provide an alternative therapeutic for B-cell lymphoma and
other diseases. The glycoengineered antibodies of the present
invention advantageously can be used to alter current routes of
administration and current therapeutic regimens, as their increased
effector function means they can be dosed at lower concentrations
and with less frequency, thereby reducing the potential for
antibody toxicity and/or development of antibody tolerance.
Furthermore, the improved effector function yields new approaches
to treating clinical indications that have previously been
resistant or refractory to treatment with the corresponding
anti-CD20 monoclonal antibody produced in recombinant host
systems.
A Method of Glycoengineering of Anti-CD20 Antibodies
The anti-CD20 glycoengineered antibodies of the invention can be
produced by Fc glycoengineering from any anti-CD20 monoclonal
antibodies ("parental antibodies"), which may be commercially
available or in the preclinical or clinical development. Fc
glycoengineering may be performed enzymatically or
chemoenzymatically. In a preferred embodiment, the parental
antibody is Rituximab.
The N-glycans in the glycoengineered antibodies of the invention
are preferably defucosylated.
Disclosed herein includes an improved method for making
glycoengineered antibodies, such as an anti-CD20 glycoengineered
antibody. A method of the invention may comprise the steps of (a)
contacting an anti-CD20 monoclonal antibody with an
.alpha.-fucosidase and endoglycosidase, thereby yielding a
defucosylated antibody having a single N-acetylglucosamine
(GlcNAc), and (b) adding a carbohydrate moiety to GlcNAc under
suitable conditions.
In some embodiments, the anti-CD20 monoclonal antibody according to
the method of the invention is Rituximab.
Any suitable endoglycosidase may be used to trim off the variable
portions of an oligosaccharide in N-glycan. Examples of
endoglycosidases used herein include EndoS2.
Step (a) in the method of the invention leads to a defucosylated
antibody having a single N-acetylglucosamine (GlcNAc).
Subsequently, EndoS2 mutants mediate transglycosylations to add a
broad range of selected carbohydrate moieties to GlcNAc to extend
the sugar chain. A homogenous population of glycoantibodies can
therefore be produced. Examples of transglycosylases as described
herein include the selected mutants of EndoS2 having the sequences
of SEQ ID NOs. 2-17.
In some embodiments, the carbohydrate moieties according to
embodiments of the invention may comprise diverse N-glycans of high
mannose, hybrid and complex types having the formula:
##STR00005## wherein, R.sup.1 is --H or N-acetyl glucosamine
attached via a .beta.-1,4 linkage and R.sup.2 and R.sup.3 are same
or different and are independently selected from the glycosyl
groups shown in FIG. 13.
In some embodiments, the carbohydrate moiety is a sugar
oxazoline.
Suitable conditions also include incubation of the reaction mixture
for at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60
minutes, 70 minutes, 80 minutes, 90 minutes or 100 minutes,
preferably less than 60 minutes. Incubation preferably takes place
at room temperature, more preferably at approximately 20.degree.
C., 25.degree. C., 30.degree. C., 35.degree. C., 40.degree. C. or
45.degree. C., and most preferably at approximately 37.degree.
C.
Pharmaceutical Formulations
Therapeutic formulations comprising an antibody of the invention
may be prepared for storage by mixing the antibody having the
desired degree of purity with one or more optional physiologically
acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the
form of aqueous solutions, lyophilized or other dried formulations.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, histidine and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
including, but not limited to, those with complementary activities
that do not adversely affect each other. Such molecules are
suitably present in combination in amounts that are effective for
the purpose intended.
The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
The formulations to be used for in vivo administration must be
sterile. This is readily accomplished by filtration through sterile
filtration membranes.
Sustained-release preparations may be prepared. Suitable examples
of sustained-release preparations include semipermeable matrices of
solid hydrophobic polymers containing the immunoglobulin of the
invention, which matrices are in the form of shaped articles, e.g.,
films, or microcapsule. Examples of sustained-release matrices
include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(--)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated immunoglobulins remain
in the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
The amount of antibody in the pre-lyophilized formulation is
determined taking into account the desired dose volumes, mode(s) of
administration etc. Where the protein of choice is an intact
antibody (a full-length antibody), from about 2 mg/mL to about 50
mg/mL, preferably from about 5 mg/mL to about 40 mg/mL and most
preferably from about 20-30 mg/mL is an exemplary starting protein
concentration. The protein is generally present in solution. For
example, the protein may be present in a pH-buffered solution at a
pH from about 4-8, and preferably from about 5-7. Exemplary buffers
include histidine, phosphate, Tris, citrate, succinate and other
organic acids. The buffer concentration can be from about 1 mM to
about 20 mM, or from about 3 mM to about 15 mM, depending, for
example, on the buffer and the desired isotonicity of the
formulation (e.g. of the reconstituted formulation). The preferred
buffer is histidine in that, as demonstrated below, this can have
lyoprotective properties. Succinate was shown to be another useful
buffer.
The lyoprotectant is added to the pre-lyophilized formulation. In
preferred embodiments, the lyoprotectant is a non-reducing sugar
such as sucrose or trehalose. The amount of lyoprotectant in the
pre-lyophilized formulation is generally such that, upon
reconstitution, the resulting formulation will be isotonic.
However, hypertonic reconstituted formulations may also be
suitable. In addition, the amount of lyoprotectant must not be too
low such that an unacceptable amount of degradation/aggregation of
the protein occurs upon lyophilization. Where the lyoprotectant is
a sugar (such as sucrose or trehalose) and the protein is an
antibody, exemplary lyoprotectant concentrations in the
pre-lyophilized formulation are from about 10 mM to about 400 mM,
and preferably from about 30 mM to about 300 mM, and most
preferably from about 50 mM to about 100 mM.
The ratio of protein to lyoprotectant is selected for each protein
and lyoprotectant combination. In the case of an antibody as the
protein of choice and a sugar (e.g., sucrose or trehalose) as the
lyoprotectant for generating an isotonic reconstituted formulation
with a high protein concentration, the molar ratio of lyoprotectant
to antibody may be from about 100 to about 1500 moles lyoprotectant
to 1 mole antibody, and preferably from about 200 to about 1000
moles of lyoprotectant to 1 mole antibody, for example from about
200 to about 600 moles of lyoprotectant to 1 mole antibody.
In preferred embodiments of the invention, it has been found to be
desirable to add a surfactant to the pre-lyophilized formulation.
Alternatively, or in addition, the surfactant may be added to the
lyophilized formulation and/or the reconstituted formulation.
Exemplary surfactants include nonionic surfactants such as
polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g.
poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel
sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or
stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or
stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;
lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,
myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine
(e.g lauroamidopropyl); myristamidopropyl palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68 etc). The amount of surfactant added is such that
it reduces aggregation of the reconstituted protein and minimizes
the formation of particulates after reconstitution. For example,
the surfactant may be present in the pre-lyophilized formulation in
an amount from about 0.001-0.5%, and preferably from about
0.005-0.05%.
In certain embodiments of the invention, a mixture of the
lyoprotectant (such as sucrose or trihalose) and a bulking agent
(e.g. mannitol or glycine) is used in the preparation of the
pre-lyophilization formulation. The bulking agent may allow for the
production of a uniform lyophilized cake without excessive pockets
therein etc.
Other pharmaceutically acceptable carriers, excipients or
stabilizers such as those described in Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980) may be included in the
pre-lyophilized formulation (and/or the lyophilized formulation
and/or the reconstituted formulation) provided that they do not
adversely affect the desired characteristics of the formulation.
Acceptable carriers, excipients or stabilizers are nontoxic to
recipients at the dosages and concentrations employed and include;
additional buffering agents; preservatives; co-solvents;
antioxidants including ascorbic acid and methionine; chelating
agents such as EDTA; metal complexes (e.g. Zn-protein complexes);
biodegradable polymers such as polyesters; and/or salt-forming
counterions such as sodium.
The pharmaceutical compositions and formulations described herein
are preferably stable. A "stable" formulation/composition is one in
which the antibody therein essentially retains its physical and
chemical stability and integrity upon storage. Various analytical
techniques for measuring protein stability are available in the art
and are reviewed in Peptide and Protein Drug Delivery, 247-301,
Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991)
and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability
can be measured at a selected temperature for a selected time
period.
The formulations to be used for in vivo administration must be
sterile. This is readily accomplished by filtration through sterile
filtration membranes, prior to, or following, lyophilization and
reconstitution. Alternatively, sterility of the entire mixture may
be accomplished by autoclaving the ingredients, except for protein,
at about 120.degree. C. for about 30 minutes, for example.
After the protein, lyoprotectant and other optional components are
mixed together, the formulation is lyophilized. Many different
freeze-dryers are available for this purpose such as Hull50.RTM.
(Hull, USA) or GT20.RTM. (Leybold-Heraeus, Germany) freeze-dryers.
Freeze-drying is accomplished by freezing the formulation and
subsequently subliming ice from the frozen content at a temperature
suitable for primary drying. Under this condition, the product
temperature is below the eutectic point or the collapse temperature
of the formulation. Typically, the shelf temperature for the
primary drying will range from about -30 to 25.degree. C. (provided
the product remains frozen during primary drying) at a suitable
pressure, ranging typically from about 50 to 250 mTorr. The
formulation, size and type of the container holding the sample
(e.g., glass vial) and the volume of liquid will mainly dictate the
time required for drying, which can range from a few hours to
several days (e.g. 40-60 hrs). A secondary drying stage may be
carried out at about 0-40.degree. C., depending primarily on the
type and size of container and the type of protein employed.
However, it was found herein that a secondary drying step may not
be necessary. For example, the shelf temperature throughout the
entire water removal phase of lyophilization may be from about
15-30.degree. C. (e.g., about 20.degree. C.). The time and pressure
required for secondary drying will be that which produces a
suitable lyophilized cake, dependent, e.g., on the temperature and
other parameters. The secondary drying time is dictated by the
desired residual moisture level in the product and typically takes
at least about 5 hours (e.g. 10-15 hours). The pressure may be the
same as that employed during the primary drying step. Freeze-drying
conditions can be varied depending on the formulation and vial
size.
In some instances, it may be desirable to lyophilize the protein
formulation in the container in which reconstitution of the protein
is to be carried out in order to avoid a transfer step. The
container in this instance may, for example, be a 3, 5, 10, 20, 50
or 100 cc vial. As a general proposition, lyophilization will
result in a lyophilized formulation in which the moisture content
thereof is less than about 5%, and preferably less than about
3%.
At the desired stage, typically when it is time to administer the
protein to the patient, the lyophilized formulation may be
reconstituted with a diluent such that the protein concentration in
the reconstituted formulation is at least 50 mg/mL, for example
from about 50 mg/mL to about 400 mg/mL, more preferably from about
80 mg/mL to about 300 mg/mL, and most preferably from about 90
mg/mL to about 150 mg/mL. Such high protein concentrations in the
reconstituted formulation are considered to be particularly useful
where subcutaneous delivery of the reconstituted formulation is
intended. However, for other routes of administration, such as
intravenous administration, lower concentrations of the protein in
the reconstituted formulation may be desired (for example from
about 5-50 mg/mL, or from about 10-40 mg/mL protein in the
reconstituted formulation). In certain embodiments, the protein
concentration in the reconstituted formulation is significantly
higher than that in the pre-lyophilized formulation. For example,
the protein concentration in the reconstituted formulation may be
about 2-40 times, preferably 3-10 times and most preferably 3-6
times (e.g. at least three fold or at least four fold) that of the
pre-lyophilized formulation.
Reconstitution generally takes place at a temperature of about
25.degree. C. to ensure complete hydration, although other
temperatures may be employed as desired. The time required for
reconstitution will depend, e.g., on the type of diluent, amount of
excipient(s) and protein. Exemplary diluents include sterile water,
bacteriostatic water for injection (BWFI), a pH buffered solution
(e.g. phosphate-buffered saline), sterile saline solution, Ringer's
solution or dextrose solution. The diluent optionally contains a
preservative. Exemplary preservatives have been described above,
with aromatic alcohols such as benzyl or phenol alcohol being the
preferred preservatives. The amount of preservative employed is
determined by assessing different preservative concentrations for
compatibility with the protein and preservative efficacy testing.
For example, if the preservative is an aromatic alcohol (such as
benzyl alcohol), it can be present in an amount from about 0.1-2.0%
and preferably from about 0.5-1.5%, but most preferably about
1.0-1.2%. Preferably, the reconstituted formulation has less than
6000 particles per vial which are >10 .mu.m in size.
Therapeutic Applications
The glycoengineered antibodies described herein may be used for
treating a patient having a cancer. The method of the treatment
comprises administering to the patient an effective amount of a
glycoengineered antibody or a pharmaceutical composition described
herein. Examples of the cancers include, but are not limited to, B
cell lymphomas, NHL, precursor B cell lymphoblastic
leukemia/lymphoma and mature B cell neoplasms, B cell chronic
lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B cell
prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell
lymphoma (MCL), follicular lymphoma (FL), low-grade,
intermediate-grade and high-grade (FL), cutaneous follicle center
lymphoma, marginal zone B cell lymphoma, MALT type marginal zone B
cell lymphoma, nodal marginal zone B cell lymphoma, splenic type
marginal zone B cell lymphoma, hairy cell leukemia, diffuse large B
cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell
myeloma, post-transplant lymphoproliferative disorder,
Waldenstrom's macroglobulinemia, and anaplastic large-cell lymphoma
(ALCL).
In certain embodiments, the cancer is B-cell lymphoma such as
non-Hodgkin's lymphoma.
Further, the glycoengineered antibodies described herein may be
used for treating a patient having an autoimmune or inflammatory
disease. The method of the treatment comprises administering to the
patient an effective amount of a glycoengineered antibody or a
pharmaceutical composition described herein. Examples of the
autoimmune or inflammatory disease include, but are not limited to,
rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus
erythematosus (SLE), Wegener's disease, inflammatory bowel disease,
idiopathic thrombocytopenic purpura (ITP), thrombotic
thrombocytopenic purpura (TTP), autoimmune thrombocytopenia,
multiple sclerosis, psoriasis, IgA nephropathy, IgM
polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus,
Reynaud's syndrome, Crohn's disease, ulcerative colitis, gastritis,
Hashimoto's thyroiditis, ankylosing spondylitis, hepatitis
C-associated cryoglobulinemic vasculitis, chronic focal
encephalitis, bullous pemphigoid, hemophilia A,
membranoproliferative glomerulnephritis, adult and juvenile
dermatomyositis, adult polymyositis, chronic urticaria, primary
biliary cirrhosis, neuromyelitis optica, Graves' dysthyroid
disease, bullous pemphigoid, membranoproliferative
glonerulonephritis, Churg-Strauss syndrome, asthma, psoriatic
arthritis, dermatitis, respiratory distress syndrome, meningitis,
encephalitits, uveitis, eczema, atherosclerosis, leukocyte adhesion
deficiency, juvenile onset diabetes, Reiter's disease, Behcet's
disease, hemolytic anemia, atopic dermatitis, Wegener's
granulomatosis, Omenn's syndrome, chronic renal failure, acute
infectious mononucleosis, HIV and herpes-associated disease,
systemic sclerosis, Sjorgen's syndrome and glomerulonephritis,
dermatomyositis, ANCA, aplastic anemia, autoimmune hemolytic anemia
(AIHA), factor VIII deficiency, hemophilia A, autoimmune
neutropenia, Castleman's syndrome, Goodpasture's syndrome, solid
organ transplant rejection, graft versus host disease (GVHD),
autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV),
bronchiolitis obliterans (non-transplant), Guillain-Barre Syndrome,
large vessel vasculitis, giant cell (Takayasu's) arteritis, medium
vessel vasculitis, Kawasaki's Disease, and polyarteritis
nodosa.
In certain embodiments, the autoimmune or inflammatory disease is
rheumatoid arthritis.
In these treatment methods, the anti-CD20 glycoengineered antibody
can be administered alone or in conjunction with a second
therapeutic agent such as a second antibody, or a chemotherapeutic
agent or an immunosuppressive agent. The second antibody can be one
that binds CD20 or a different B cell antigen, or a NK or T cell
antigen.
Embodiments of the invention will be further illustrated with the
following specific examples. One skilled in the art would
appreciate that these specific examples are for illustration only
and that other modifications and variations are possible without
departing from the scope of the invention. For example, the EndoS2
mutants of the invention may be used to glycoengineer any
glycoproteins or glycopeptides, including antibodies. The specific
examples described herein use anti-CD20 antibodies. However, one
skilled in the art would appreciate that other glycoproteins or
antibodies may also be used in a similar manner.
Material and Methods
Monoclonal antibody Rituximab was purchased commercial source or
in-house produced. N-glycans of high mannose, hybrid and complex
type were synthesized according to previously reported procedures.
(20, 21).
Clone Constructions, Overexpression, and Purification of EndoS2 and
Mutants
The EndoS2 encoding gene, ndoS2, from Streptococcus pyogenes GAS
NZ131, was synthesized and subcloned into the pET28a expression
vector. The signal peptide sequence (amino acid 1-36) of ndoS2 was
replaced by a His.sub.6-tag on its N-terminal. The mutants of ndoS2
were generated by site-directed mutagenesis according to the
manufacturer's instructions (Agilent Technologies) that PCR
reactions were performed by using ndoS2 expression vector as a
template and oligonucleotide pairs containing desired mutation as
primers. Then, the amplified DNA was treated with DpnI and
transformed into DH5a competent cells. The mutated sequences were
confirmed by DNA sequencing (Genomics). After the transformation
into BL21 (DE3) competent cells for expression, cells were induced
with 0.1 mM isopropyl-.beta.-D-thiogalactopyranoside (IPTG), the
recombinant EndoS2 mutant proteins with their His.sub.6 tag were
expressed at 20.degree. C. for 16 h and pelleted by centrifugation
at 6500 rpm for 30 min. The cells were resuspended in lysis buffer
(30 mM HEPES, pH 8.0, 300 mM NaCl) and disrupted using Ultrasonic
Processor (10 min, 4 s-on/5 s-off, ChromTech). The total cell
lysates were centrifuged at 10000 rpm for 45 min and the soluble
recombinant EndoS2 and mutant proteins were purified by immobilized
metal-ion chromatography with a Ni-NTA column (GE Healthcare). The
eluted protein fractions were collected and concentrated against
storage buffer (30 mM HEPES, pH 8.0, 100 mM NaCl) by using Amicon
ultra centrifugal filters 10 kDa. Concentrated protein samples were
analyzed by SDS-PAGE, and protein concentration was quantified
using a Nano-Drop 2000c spectrophotometer. The yield of
overproduction of the wild-type EndoS2 was approximately 35 mg/L,
and the yield for the mutants was approximately 25 mg/L.
Preparation of GlcNAc-Rituximab from Commercially Available
Rituximab
Rituximab (2.5 mg, from commercial source) in 1.25 ml 50 mM sodium
phosphate buffer pH 7.0 was incubated with EndoS2 (125 .beta.g) and
fucosidase (2.5 mg) at 37.degree. C. for 22 h. After the complete
cleavage of the N-glycans on the heavy chain, as checked by
SDS-PAGE analyses, the reaction mixture was then subjected to
affinity chromatography on a column of 1 ml protein A-agarose resin
preequilibrated with 20 mM sodium phosphate pH 7.0. After washing,
the bound IgG was eluted with 10 ml 50 mM glycine HCl pH3.0. The
elution fractions were immediately neutralized with 1 M Tris-Cl pH
8.3 and concentrated by centrifugal filtration (Amicon Ultra
centrifugal filter) to give GlcNAc-Rituximab (1.93 mg). The product
was trypsinized, and the glycopeptides, TKPREEQYNSTYR and EEQYNSTYR
were analyzed using nanospray LC/MS to confirm the glycosylation
pattern of GlcNAc-Rituximab.
Preparation of Glycan-Oxazolines
A solution of respective glycan (3-5 mg),
2-chloro-1,3-dimethylimidazolinium chloride (DMC) (6-10 mg) and
Et.sub.3N (10-20 .mu.L) in water (300-500 .mu.L) was stirred at
4.degree. C. for 1 h. The reaction mixture was subjected to gel
filtration chromatography on a Sephadex G-25 column eluted by 0.05%
aqueous Et.sub.3N. The fractions containing the products G1-G16
(glycan oxazolines) were combined and lyophilized to give a white
powder (2.5-4 mg, Yield .about.80-90%).
Hydrolytic Activity Assay of EndoS2 and its Mutants
A solution containing 52 .mu.M rituximab (400 .mu.g) and 52 nM
EndoS2 or its selected mutant proteins in 100 mM HEPES buffer pH
7.0 was incubated at 37.degree. C. with 700 rpm shaking. At the
indicated time points, 2 .mu.g aliquots were taken and analyzed by
10% SDS-PAGE. Rituximab with glycan hydrolyzed would display faster
migration on PAGE. The relative percentage of hydrolyzed product
was calculated by using Image J software based on the intensity of
bands on SDS-PAGE (FIG. 3).
Transglycosylation Activity Assay of EndoS2 and its Mutants Using
SCT-Oxazoline as Donor Substrate
A solution containing 67.5 .mu.M mono GlcNAc-Rituximab (400 .mu.g)
and 2.5 mM sialylated complex type glycan (SCT)-oxazoline (200
.mu.g) in 100 mM HEPES buffer pH 7.0 was incubated with 67.5 nM
EndoS2 or selected mutant proteins at 37.degree. C. with 700 rpm
shaking. At the indicated time points, 2 .mu.g aliquots were taken
and analyzed by 10% SDS-PAGE. Glycosylated Rituximab would display
slower migration on PAGE. The relative percentage of Rituximab-SCT
was calculated by using Image J software based on the intensity of
bands on SDS-PAGE.
Transglycosylation of GlcNAc-Rituximab with Various Glycan
Oxazoline Using EndoS2 Mutants
General procedure: To a mixture of EndoS2 mutants (16.8 .mu.M) and
GlcNAc-Rituximab (2 mg, 0.337 mM) in 100 mM HEPES buffer (pH 7.0)
was added glycan oxazoline (2-3 mg) and incubated at 37.degree. C.
with 700 rpm shaking for 1 to 2 h. The reaction was quenched by
adding 0.1 mM of EDTA solution. The reaction mixture was purified
with protein A affinity column, followed by amanion exchange column
capto Q to collect the desired product, anti-CD20 glycoengineered
Rituximab. The product was trypsinized, and the glycopeptides,
TKPREEQYNSTYR and EEQYNSTYR, were analyzed using nanospray LC/MS to
confirm the desired glycosylation pattern.
Table 7 lists the optimized reaction details for preparation of
each of the Rituximab glycoforms
TABLE-US-00007 TABLE 7 Starting Rituximab EndoS2 Enzyme Glycan
Glycan Reaction Material Glycoform mutants ratio oxazoline amount
time GlcNAc- Rtx-G1 D226Q 16.8 .mu.M G1 2 mg 30 min Rituximab
Rtx-G2 T138Q 33.7 .mu.M G2 3 mg 30 min (2 mg, Rtx-G3 D182Q 16.8
.mu.M G3 2 mg 30 min 0.337 mM) Rtx-G4 D226Q 16.8 .mu.M G4 2 mg 60
min Rtx-G5 D182Q 16.8 .mu.M G5 3 mg 30 min Rtx-G6 D138Q 33.7 .mu.M
G6 3 mg 30 min Rtx-G7 T138Q 16.8 .mu.M G7 2 mg 30 min Rtx-G8 T138Q
16.8 .mu.M G8 2 mg 30 min Rtx-G9 D182Q 16.8 .mu.M G9 2 mg 30 min
Rtx-G10 D182Q 16.8 .mu.M G10 2 mg 30 min Rtx-G11 D182Q 16.8 .mu.M
G11 2 mg 60 min Rtx-G12 D182Q 16.8 .mu.M G12 2 mg 60 min Rtx-G13
D138Q 16.8 .mu.M G13 2 mg 30 min Rtx-G14 D182Q 16.8 .mu.M G14 3 mg
60 min Rtx-G15 D182Q 16.8 .mu.M G15 2 mg 60 min Rtx-G16 D182Q 16.8
.mu.M G15 2 mg 60 min
Binding Affinity of Glycoenginnered Anti-CD20 Antibodies to
Fc.gamma.RIIIA
The affinity of the remodeled glycoforms of Rituximab for
Fc.gamma.IIA receptors was examined by ELISA.
Microtiter plate (Corning.RTM. 96 Well Clear Flat Bottom
Polystyrene High Bind, #9018) was coated with 50 ng/well of
recombinant soluble Fc.gamma.RIIIA diluted in 50 mM
Bicarbonate/carbonate coating buffer (pH 10) overnight at 4.degree.
C. The plate was then washed 3 times with PBST (0.05% Tween 20 in
PBS) and blocked with 5% BSA in PBST for 1 h at room temperature.
The binding activity of glycoengineered antibodies (Rtx G1-G16) was
determined for serial eight dilutions, starting with concentration
of 100 .mu.g/ml in 2% BSA/PBST in duplicates. The plate was
incubated for 1 h at room temperature, and washed 3 times with
PBST. Next, 100 .mu.l of goat anti-human IgG conjugated to
horseradish peroxidase (Jackson immune, #109-035-088) in 2%
BSA/PBST was added per well and incubated for 30 min at room
temperature. The plate was washed 5 times with PBST then 100 .mu.L
per well of TMB substrate (eBioscience, #00-4201-56) was added and
the resulting plate was incubated in the dark 15 min at room
temperature. The absorbance value was determined at 450 nm in an
ELISA reader (Molecular Devices Corporation, Sunnyvale, Calif.,
USA).
ADCC Activity of Glycoenginnered Anti-CD20 Antibodies
The antibody-mediated ADCC was evaluated using the calcein release
assay. Raji cells (human Burkitt's lymphoma cell line) were
obtained from BCRC as target cells. Peripheral blood-mononuclear
cells (PBMC) were separated from the blood of healthy volunteers
using Ficoll-Paque (GE healthcare) as effector cells. The target
cells (1.times.10.sup.6/ml) were labeled for 30 minutes at
37.degree. C. with 10 .mu.M calcein-acetoxymethyl ester (Thermo
Fisher Scientific). After washing, labeled target cells were
distributed in 96-well plates at a density of 1.times.10.sup.4
cells in 50 .mu.l per well. Antibodies with various concentrations,
and effector cells (2.5.times.10.sup.5 per well) with a 25:1 E/T
ratio were then added. After incubation for 4 h at 37.degree. C.,
cells were sedimented by centrifugation, and 150 .mu.lof
supernatants were collected and analyzed by using fluorescence
microplate reader to measure the release of calcein. For maximal
release, the cells were lysed with 1% Triton X-100. The
fluorescence value of the culture medium background was subtracted
from that of the experimental results.
While the invention has been described with respect to a limited
number of embodiments, those skilled in the art, having benefit of
this disclosure, will appreciate that other embodiments can be
devised which do not depart from the scope of the invention as
disclosed herein. Accordingly, the scope of the invention should be
limited only by the attached claims.
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Glycosylation as a strategy to improve antibody-based therapeutics.
Nat. Rev. Drug Discov. 2009, 8(3), 226-34. 16. Wang L. X.,
Chemoenzymatic Glycoengineering of Intact IgG Antibodies for Gain
of Functions. J. Am. Chem. Soc. 2012, 134(29), 12308-12318. 17.
Collin M.; Olsen A., EndoS, a novel secreted protein from
Streptococcus pyogenes with endoglycosidase activity on human IgG.
EMBO J. 2001, 20(12), 3046-55. 18. Sjogren J; Struwe W. B.;
Cosgrave E. F.; Rudd P. M.; Stervander M.; Allhorn M.; Hollands A.;
Nizet V.; Collin M., EndoS2 is a unique and conserved enzyme of
serotype M49 group A Streptococcus that hydrolyses N-linked glycans
on IgG and .alpha.1-acid glycoprotein. Biochem J. 2013, 455(1),
107-18. 19. Sjogren J.; Collin M., EndoS and EndoS2 hydrolyze
Fc-glycans on therapeutic antibodies with different glycoform
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high-mannose glycans. Glycobiology. 2015, 25(10), 1053-1063. 20.
Shivatare, S. S., Wu, C. Y., Wong, C., H, Efficient convergent
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SEQUENCE LISTINGS
1
191842PRTStreptococcus pyogenes 1Met Asp Lys His Leu Leu Val Lys
Arg Thr Leu Gly Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly Ala Ala
Leu Ala Thr His His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala Glu Glu
Lys Thr Val Gln Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly Ala Lys
Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu Tyr Ala
Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr65 70 75 80Gly Ile
Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val 85 90 95Pro
Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser 100 105
110Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn
Glu Leu 130 135 140Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp
Thr Pro Glu Gly145 150 155 160Asn Lys Ala Leu Ala Ala Ala Ile Val
Lys Ala Phe Val Thr Asp Arg 165 170 175Gly Val Asp Gly Leu Asp Ile
Asp Ile Glu His Glu Phe Thr Asn Lys 180 185 190Arg Thr Pro Glu Glu
Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile 195 200 205Ala Gln Leu
Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile 210 215 220Met
Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile225 230
235 240Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln
Gly 245 250 255Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn
Gln Tyr Gln 260 265 270Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly
Phe Ser Phe Phe Glu 275 280 285Glu Ser Ala Ser Lys Gly Asn Leu Trp
Phe Asp Val Asn Glu Tyr Asp 290 295 300Pro Asn Asn Pro Glu Lys Gly
Lys Asp Ile Glu Gly Thr Arg Ala Lys305 310 315 320Lys Tyr Ala Glu
Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile 325 330 335Phe Ser
Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr 340 345
350Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr
Leu Met 370 375 380Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys
Asp Ile Pro Asp385 390 395 400Pro Ala Leu Arg Glu Gln Ile Ile Gln
Gln Val Gly Gln Tyr Lys Gly 405 410 415Asp Leu Glu Arg Tyr Asn Lys
Thr Leu Val Leu Thr Gly Asp Lys Ile 420 425 430Gln Asn Leu Lys Gly
Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu 435 440 445Leu Arg Gln
Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro 450 455 460Glu
Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly465 470
475 480Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp
Gly 485 490 495Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp
Ile Ser His 500 505 510Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp
Arg Lys Leu Leu Met 515 520 525Thr Leu Met Glu Gln Val Ser Asn His
Gln Lys Ile Thr Val Lys Asn 530 535 540Thr Ala Phe Glu Asn Gln Lys
Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr545 550 555 560Asp Thr Lys Glu
Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile 565 570 575Leu Thr
Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile 580 585
590Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg
Lys Asp 610 615 620Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn
Leu Gly Glu Thr625 630 635 640Val Thr Ser Lys Val Thr Ala Thr Thr
Asp Glu Thr Tyr Leu Val Asp 645 650 655Val Ser Asp Gly Glu Lys Val
Val His His Met Lys Leu Asn Ile Gly 660 665 670Ser Gly Ala Ile Met
Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile 675 680 685Gly Thr Ser
Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu 690 695 700Lys
Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe705 710
715 720Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn
Ala 725 730 735Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn
Val Ala Lys 740 745 750Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile
Asp Leu Glu Lys Met 755 760 765Asp Ile Lys Asn Arg Lys Glu Tyr Leu
Ser Asn Asp Glu Asn Trp Thr 770 775 780Asp Val Ala Gln Met Asp Asp
Ala Lys Ala Ile Phe Asn Ser Lys Leu785 790 795 800Ser Asn Val Leu
Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala 805 810 815Ser Ser
Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg 820 825
830Leu Ser Asn Asp Val Ala Asn Thr Leu Asp 835 8402842PRTArtificial
SequenceSynthetic 2Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly
Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His
His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala Glu Glu Lys Thr Val Gln
Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly Ala Lys Leu Val Gln Glu
Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg
Thr Trp His Asp Arg Ala Ser Thr65 70 75 80Gly Ile Asp Gly Lys Gln
Gln His Pro Glu Asn Thr Met Ala Glu Val 85 90 95Pro Lys Glu Val Asp
Ile Leu Phe Val Phe His Asp His Thr Ala Ser 100 105 110Asp Ser Pro
Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu 115 120 125His
Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu 130 135
140Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu
Gly145 150 155 160Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe
Val Thr Asp Arg 165 170 175Gly Val Asp Gly Leu Gln Ile Asp Ile Glu
His Glu Phe Thr Asn Lys 180 185 190Arg Thr Pro Glu Glu Asp Ala Arg
Ala Leu Asn Val Phe Lys Glu Ile 195 200 205Ala Gln Leu Ile Gly Lys
Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile 210 215 220Met Asp Thr Thr
Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile225 230 235 240Ala
Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly 245 250
255Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe
Phe Glu 275 280 285Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val
Asn Glu Tyr Asp 290 295 300Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile
Glu Gly Thr Arg Ala Lys305 310 315 320Lys Tyr Ala Glu Trp Gln Pro
Ser Thr Gly Gly Leu Lys Ala Gly Ile 325 330 335Phe Ser Tyr Ala Ile
Asp Arg Asp Gly Val Ala His Val Pro Ser Thr 340 345 350Tyr Lys Asn
Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn 355 360 365Ile
Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met 370 375
380Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro
Asp385 390 395 400Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly
Gln Tyr Lys Gly 405 410 415Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val
Leu Thr Gly Asp Lys Ile 420 425 430Gln Asn Leu Lys Gly Leu Glu Lys
Leu Ser Lys Leu Gln Lys Leu Glu 435 440 445Leu Arg Gln Leu Ser Asn
Val Lys Glu Ile Thr Pro Glu Leu Leu Pro 450 455 460Glu Ser Met Lys
Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly465 470 475 480Leu
Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly 485 490
495Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu
Leu Met 515 520 525Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile
Thr Val Lys Asn 530 535 540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly
Tyr Tyr Pro Gln Thr Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr
Asp Val Asp Asn Ala Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val
Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu
Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg
Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615
620Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu
Thr625 630 635 640Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr
Tyr Leu Val Asp 645 650 655Val Ser Asp Gly Glu Lys Val Val His His
Met Lys Leu Asn Ile Gly 660 665 670Ser Gly Ala Ile Met Met Glu Asn
Leu Ala Lys Gly Ala Lys Val Ile 675 680 685Gly Thr Ser Gly Asp Phe
Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg
Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe705 710 715 720Asp
Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730
735Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu
Lys Met 755 760 765Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp
Glu Asn Trp Thr 770 775 780Asp Val Ala Gln Met Asp Asp Ala Lys Ala
Ile Phe Asn Ser Lys Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr
Trp Arg Phe Cys Val Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro
Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn
Asp Val Ala Asn Thr Leu Asp 835 8403842PRTArtificial
SequenceSynthetic 3Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly
Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His
His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala Glu Glu Lys Thr Val Gln
Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly Ala Lys Leu Val Gln Glu
Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg
Thr Trp His Asp Arg Ala Ser Thr65 70 75 80Gly Ile Asp Gly Lys Gln
Gln His Pro Glu Asn Thr Met Ala Glu Val 85 90 95Pro Lys Glu Val Asp
Ile Leu Phe Val Phe His Asp His Thr Ala Ser 100 105 110Asp Ser Pro
Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu 115 120 125His
Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu 130 135
140Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu
Gly145 150 155 160Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe
Val Thr Asp Arg 165 170 175Gly Val Asp Gly Leu Asp Ile Asp Ile Glu
His Glu Phe Thr Asn Lys 180 185 190Arg Thr Pro Glu Glu Asp Ala Arg
Ala Leu Asn Val Phe Lys Glu Ile 195 200 205Ala Gln Leu Ile Gly Lys
Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile 210 215 220Met Gln Thr Thr
Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile225 230 235 240Ala
Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly 245 250
255Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe
Phe Glu 275 280 285Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val
Asn Glu Tyr Asp 290 295 300Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile
Glu Gly Thr Arg Ala Lys305 310 315 320Lys Tyr Ala Glu Trp Gln Pro
Ser Thr Gly Gly Leu Lys Ala Gly Ile 325 330 335Phe Ser Tyr Ala Ile
Asp Arg Asp Gly Val Ala His Val Pro Ser Thr 340 345 350Tyr Lys Asn
Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn 355 360 365Ile
Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met 370 375
380Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro
Asp385 390 395 400Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly
Gln Tyr Lys Gly 405 410 415Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val
Leu Thr Gly Asp Lys Ile 420 425 430Gln Asn Leu Lys Gly Leu Glu Lys
Leu Ser Lys Leu Gln Lys Leu Glu 435 440 445Leu Arg Gln Leu Ser Asn
Val Lys Glu Ile Thr Pro Glu Leu Leu Pro 450 455 460Glu Ser Met Lys
Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly465 470 475 480Leu
Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly 485 490
495Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu
Leu Met 515 520 525Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile
Thr Val Lys Asn 530 535 540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly
Tyr Tyr Pro Gln Thr Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr
Asp Val Asp Asn Ala Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val
Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu
Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg
Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615
620Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu
Thr625 630 635 640Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr
Tyr Leu Val Asp 645 650 655Val Ser Asp Gly Glu Lys Val Val His His
Met Lys Leu Asn Ile Gly 660 665 670Ser Gly Ala Ile Met Met Glu Asn
Leu Ala Lys Gly Ala Lys Val Ile 675 680 685Gly Thr Ser Gly Asp Phe
Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg
Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe705 710 715 720Asp
Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730
735Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu
Lys Met 755 760 765Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn
Asp
Glu Asn Trp Thr 770 775 780Asp Val Ala Gln Met Asp Asp Ala Lys Ala
Ile Phe Asn Ser Lys Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr
Trp Arg Phe Cys Val Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro
Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn
Asp Val Ala Asn Thr Leu Asp 835 8404842PRTArtificial
SequenceSynthetic 4Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly
Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His
His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala Glu Glu Lys Thr Val Gln
Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly Ala Lys Leu Val Gln Glu
Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg
Thr Trp His Asp Arg Ala Ser Thr65 70 75 80Gly Ile Asp Gly Lys Gln
Gln His Pro Glu Asn Thr Met Ala Glu Val 85 90 95Pro Lys Glu Val Asp
Ile Leu Phe Val Phe His Asp His Thr Ala Ser 100 105 110Asp Ser Pro
Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu 115 120 125His
Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu 130 135
140Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu
Gly145 150 155 160Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe
Val Thr Asp Arg 165 170 175Gly Val Asp Gly Leu Asp Ile Asp Ile Glu
His Glu Phe Thr Asn Lys 180 185 190Arg Thr Pro Glu Glu Asp Ala Arg
Ala Leu Asn Val Phe Lys Glu Ile 195 200 205Ala Gln Leu Ile Gly Lys
Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile 210 215 220Met Asp Gln Thr
Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile225 230 235 240Ala
Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly 245 250
255Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe
Phe Glu 275 280 285Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val
Asn Glu Tyr Asp 290 295 300Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile
Glu Gly Thr Arg Ala Lys305 310 315 320Lys Tyr Ala Glu Trp Gln Pro
Ser Thr Gly Gly Leu Lys Ala Gly Ile 325 330 335Phe Ser Tyr Ala Ile
Asp Arg Asp Gly Val Ala His Val Pro Ser Thr 340 345 350Tyr Lys Asn
Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn 355 360 365Ile
Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met 370 375
380Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro
Asp385 390 395 400Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly
Gln Tyr Lys Gly 405 410 415Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val
Leu Thr Gly Asp Lys Ile 420 425 430Gln Asn Leu Lys Gly Leu Glu Lys
Leu Ser Lys Leu Gln Lys Leu Glu 435 440 445Leu Arg Gln Leu Ser Asn
Val Lys Glu Ile Thr Pro Glu Leu Leu Pro 450 455 460Glu Ser Met Lys
Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly465 470 475 480Leu
Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly 485 490
495Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu
Leu Met 515 520 525Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile
Thr Val Lys Asn 530 535 540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly
Tyr Tyr Pro Gln Thr Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr
Asp Val Asp Asn Ala Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val
Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu
Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg
Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615
620Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu
Thr625 630 635 640Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr
Tyr Leu Val Asp 645 650 655Val Ser Asp Gly Glu Lys Val Val His His
Met Lys Leu Asn Ile Gly 660 665 670Ser Gly Ala Ile Met Met Glu Asn
Leu Ala Lys Gly Ala Lys Val Ile 675 680 685Gly Thr Ser Gly Asp Phe
Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg
Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe705 710 715 720Asp
Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730
735Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu
Lys Met 755 760 765Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp
Glu Asn Trp Thr 770 775 780Asp Val Ala Gln Met Asp Asp Ala Lys Ala
Ile Phe Asn Ser Lys Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr
Trp Arg Phe Cys Val Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro
Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn
Asp Val Ala Asn Thr Leu Asp 835 8405842PRTArtificial
SequenceSynthetic 5Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly
Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His
His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala Glu Glu Lys Thr Val Gln
Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly Ala Lys Leu Val Gln Glu
Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg
Thr Trp His Asp Arg Ala Ser Thr65 70 75 80Gly Ile Asp Gly Lys Gln
Gln His Pro Glu Asn Thr Met Ala Glu Val 85 90 95Pro Lys Glu Val Asp
Ile Leu Phe Val Phe His Asp His Thr Ala Ser 100 105 110Asp Ser Pro
Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu 115 120 125His
Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu 130 135
140Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu
Gly145 150 155 160Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe
Val Thr Asp Arg 165 170 175Gly Val Asp Gly Leu Asp Ile Asp Ile Glu
His Glu Phe Thr Asn Lys 180 185 190Arg Thr Pro Glu Glu Asp Ala Arg
Ala Leu Asn Val Phe Lys Glu Ile 195 200 205Ala Gln Leu Ile Gly Lys
Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile 210 215 220Met Asp Thr Gln
Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile225 230 235 240Ala
Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly 245 250
255Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe
Phe Glu 275 280 285Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val
Asn Glu Tyr Asp 290 295 300Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile
Glu Gly Thr Arg Ala Lys305 310 315 320Lys Tyr Ala Glu Trp Gln Pro
Ser Thr Gly Gly Leu Lys Ala Gly Ile 325 330 335Phe Ser Tyr Ala Ile
Asp Arg Asp Gly Val Ala His Val Pro Ser Thr 340 345 350Tyr Lys Asn
Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn 355 360 365Ile
Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met 370 375
380Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro
Asp385 390 395 400Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly
Gln Tyr Lys Gly 405 410 415Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val
Leu Thr Gly Asp Lys Ile 420 425 430Gln Asn Leu Lys Gly Leu Glu Lys
Leu Ser Lys Leu Gln Lys Leu Glu 435 440 445Leu Arg Gln Leu Ser Asn
Val Lys Glu Ile Thr Pro Glu Leu Leu Pro 450 455 460Glu Ser Met Lys
Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly465 470 475 480Leu
Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly 485 490
495Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu
Leu Met 515 520 525Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile
Thr Val Lys Asn 530 535 540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly
Tyr Tyr Pro Gln Thr Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr
Asp Val Asp Asn Ala Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val
Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu
Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg
Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615
620Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu
Thr625 630 635 640Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr
Tyr Leu Val Asp 645 650 655Val Ser Asp Gly Glu Lys Val Val His His
Met Lys Leu Asn Ile Gly 660 665 670Ser Gly Ala Ile Met Met Glu Asn
Leu Ala Lys Gly Ala Lys Val Ile 675 680 685Gly Thr Ser Gly Asp Phe
Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg
Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe705 710 715 720Asp
Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730
735Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu
Lys Met 755 760 765Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp
Glu Asn Trp Thr 770 775 780Asp Val Ala Gln Met Asp Asp Ala Lys Ala
Ile Phe Asn Ser Lys Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr
Trp Arg Phe Cys Val Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro
Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn
Asp Val Ala Asn Thr Leu Asp 835 8406842PRTArtificial
SequenceSynthetic 6Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly
Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His
His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala Glu Glu Lys Thr Val Gln
Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly Ala Lys Leu Val Gln Glu
Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg
Thr Trp His Asp Arg Ala Ser Thr65 70 75 80Gly Ile Asp Gly Lys Gln
Gln His Pro Glu Asn Thr Met Ala Glu Val 85 90 95Pro Lys Glu Val Asp
Ile Leu Phe Val Phe His Asp His Thr Ala Ser 100 105 110Asp Ser Pro
Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu 115 120 125His
Gln Gln Gly Thr Ala Leu Val Gln Asp Ile Gly Val Asn Glu Leu 130 135
140Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu
Gly145 150 155 160Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe
Val Thr Asp Arg 165 170 175Gly Val Asp Gly Leu Asp Ile Asp Ile Glu
His Glu Phe Thr Asn Lys 180 185 190Arg Thr Pro Glu Glu Asp Ala Arg
Ala Leu Asn Val Phe Lys Glu Ile 195 200 205Ala Gln Leu Ile Gly Lys
Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile 210 215 220Met Asp Thr Thr
Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile225 230 235 240Ala
Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly 245 250
255Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe
Phe Glu 275 280 285Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val
Asn Glu Tyr Asp 290 295 300Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile
Glu Gly Thr Arg Ala Lys305 310 315 320Lys Tyr Ala Glu Trp Gln Pro
Ser Thr Gly Gly Leu Lys Ala Gly Ile 325 330 335Phe Ser Tyr Ala Ile
Asp Arg Asp Gly Val Ala His Val Pro Ser Thr 340 345 350Tyr Lys Asn
Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn 355 360 365Ile
Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met 370 375
380Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro
Asp385 390 395 400Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly
Gln Tyr Lys Gly 405 410 415Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val
Leu Thr Gly Asp Lys Ile 420 425 430Gln Asn Leu Lys Gly Leu Glu Lys
Leu Ser Lys Leu Gln Lys Leu Glu 435 440 445Leu Arg Gln Leu Ser Asn
Val Lys Glu Ile Thr Pro Glu Leu Leu Pro 450 455 460Glu Ser Met Lys
Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly465 470 475 480Leu
Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly 485 490
495Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu
Leu Met 515 520 525Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile
Thr Val Lys Asn 530 535 540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly
Tyr Tyr Pro Gln Thr Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr
Asp Val Asp Asn Ala Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val
Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu
Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg
Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615
620Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu
Thr625 630 635 640Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr
Tyr Leu Val Asp 645 650 655Val Ser Asp Gly Glu Lys Val Val His His
Met Lys Leu Asn Ile Gly 660 665 670Ser Gly Ala Ile Met Met Glu Asn
Leu Ala Lys Gly Ala Lys Val Ile 675 680 685Gly Thr Ser Gly Asp Phe
Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg
Phe
Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe705 710 715 720Asp Leu
Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730
735Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu
Lys Met 755 760 765Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp
Glu Asn Trp Thr 770 775 780Asp Val Ala Gln Met Asp Asp Ala Lys Ala
Ile Phe Asn Ser Lys Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr
Trp Arg Phe Cys Val Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro
Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn
Asp Val Ala Asn Thr Leu Asp 835 8407842PRTArtificial
SequenceSynthetic 7Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly
Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His
His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala Glu Glu Lys Thr Val Gln
Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly Ala Lys Leu Val Gln Glu
Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg
Thr Trp His Asp Arg Ala Ser Thr65 70 75 80Gly Ile Asp Gly Lys Gln
Gln His Pro Glu Asn Thr Met Ala Glu Val 85 90 95Pro Lys Glu Val Asp
Ile Leu Phe Val Phe His Asp His Thr Ala Ser 100 105 110Asp Ser Pro
Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu 115 120 125His
Gln Gln Gly Thr Ala Leu Val Gln Glu Ile Gly Val Asn Glu Leu 130 135
140Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu
Gly145 150 155 160Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe
Val Thr Asp Arg 165 170 175Gly Val Asp Gly Leu Asp Ile Asp Ile Glu
His Glu Phe Thr Asn Lys 180 185 190Arg Thr Pro Glu Glu Asp Ala Arg
Ala Leu Asn Val Phe Lys Glu Ile 195 200 205Ala Gln Leu Ile Gly Lys
Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile 210 215 220Met Asp Thr Thr
Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile225 230 235 240Ala
Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly 245 250
255Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe
Phe Glu 275 280 285Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val
Asn Glu Tyr Asp 290 295 300Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile
Glu Gly Thr Arg Ala Lys305 310 315 320Lys Tyr Ala Glu Trp Gln Pro
Ser Thr Gly Gly Leu Lys Ala Gly Ile 325 330 335Phe Ser Tyr Ala Ile
Asp Arg Asp Gly Val Ala His Val Pro Ser Thr 340 345 350Tyr Lys Asn
Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn 355 360 365Ile
Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met 370 375
380Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro
Asp385 390 395 400Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly
Gln Tyr Lys Gly 405 410 415Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val
Leu Thr Gly Asp Lys Ile 420 425 430Gln Asn Leu Lys Gly Leu Glu Lys
Leu Ser Lys Leu Gln Lys Leu Glu 435 440 445Leu Arg Gln Leu Ser Asn
Val Lys Glu Ile Thr Pro Glu Leu Leu Pro 450 455 460Glu Ser Met Lys
Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly465 470 475 480Leu
Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly 485 490
495Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu
Leu Met 515 520 525Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile
Thr Val Lys Asn 530 535 540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly
Tyr Tyr Pro Gln Thr Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr
Asp Val Asp Asn Ala Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val
Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu
Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg
Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615
620Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu
Thr625 630 635 640Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr
Tyr Leu Val Asp 645 650 655Val Ser Asp Gly Glu Lys Val Val His His
Met Lys Leu Asn Ile Gly 660 665 670Ser Gly Ala Ile Met Met Glu Asn
Leu Ala Lys Gly Ala Lys Val Ile 675 680 685Gly Thr Ser Gly Asp Phe
Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg
Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe705 710 715 720Asp
Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730
735Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu
Lys Met 755 760 765Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp
Glu Asn Trp Thr 770 775 780Asp Val Ala Gln Met Asp Asp Ala Lys Ala
Ile Phe Asn Ser Lys Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr
Trp Arg Phe Cys Val Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro
Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn
Asp Val Ala Asn Thr Leu Asp 835 8408842PRTArtificial
SequenceSynthetic 8Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly
Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His
His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala Glu Glu Lys Thr Val Gln
Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly Ala Lys Leu Val Gln Glu
Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg
Thr Trp His Asp Arg Ala Ser Thr65 70 75 80Gly Ile Asp Gly Lys Gln
Gln His Pro Glu Asn Thr Met Ala Glu Val 85 90 95Pro Lys Glu Val Asp
Ile Leu Phe Val Phe His Asp His Thr Ala Ser 100 105 110Asp Ser Pro
Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu 115 120 125His
Gln Gln Gly Thr Ala Leu Val Gln Phe Ile Gly Val Asn Glu Leu 130 135
140Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu
Gly145 150 155 160Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe
Val Thr Asp Arg 165 170 175Gly Val Asp Gly Leu Asp Ile Asp Ile Glu
His Glu Phe Thr Asn Lys 180 185 190Arg Thr Pro Glu Glu Asp Ala Arg
Ala Leu Asn Val Phe Lys Glu Ile 195 200 205Ala Gln Leu Ile Gly Lys
Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile 210 215 220Met Asp Thr Thr
Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile225 230 235 240Ala
Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly 245 250
255Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe
Phe Glu 275 280 285Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val
Asn Glu Tyr Asp 290 295 300Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile
Glu Gly Thr Arg Ala Lys305 310 315 320Lys Tyr Ala Glu Trp Gln Pro
Ser Thr Gly Gly Leu Lys Ala Gly Ile 325 330 335Phe Ser Tyr Ala Ile
Asp Arg Asp Gly Val Ala His Val Pro Ser Thr 340 345 350Tyr Lys Asn
Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn 355 360 365Ile
Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met 370 375
380Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro
Asp385 390 395 400Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly
Gln Tyr Lys Gly 405 410 415Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val
Leu Thr Gly Asp Lys Ile 420 425 430Gln Asn Leu Lys Gly Leu Glu Lys
Leu Ser Lys Leu Gln Lys Leu Glu 435 440 445Leu Arg Gln Leu Ser Asn
Val Lys Glu Ile Thr Pro Glu Leu Leu Pro 450 455 460Glu Ser Met Lys
Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly465 470 475 480Leu
Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly 485 490
495Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu
Leu Met 515 520 525Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile
Thr Val Lys Asn 530 535 540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly
Tyr Tyr Pro Gln Thr Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr
Asp Val Asp Asn Ala Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val
Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu
Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg
Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615
620Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu
Thr625 630 635 640Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr
Tyr Leu Val Asp 645 650 655Val Ser Asp Gly Glu Lys Val Val His His
Met Lys Leu Asn Ile Gly 660 665 670Ser Gly Ala Ile Met Met Glu Asn
Leu Ala Lys Gly Ala Lys Val Ile 675 680 685Gly Thr Ser Gly Asp Phe
Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg
Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe705 710 715 720Asp
Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730
735Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu
Lys Met 755 760 765Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp
Glu Asn Trp Thr 770 775 780Asp Val Ala Gln Met Asp Asp Ala Lys Ala
Ile Phe Asn Ser Lys Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr
Trp Arg Phe Cys Val Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro
Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn
Asp Val Ala Asn Thr Leu Asp 835 8409842PRTArtificial
SequenceSynthetic 9Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly
Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His
His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala Glu Glu Lys Thr Val Gln
Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly Ala Lys Leu Val Gln Glu
Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg
Thr Trp His Asp Arg Ala Ser Thr65 70 75 80Gly Ile Asp Gly Lys Gln
Gln His Pro Glu Asn Thr Met Ala Glu Val 85 90 95Pro Lys Glu Val Asp
Ile Leu Phe Val Phe His Asp His Thr Ala Ser 100 105 110Asp Ser Pro
Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu 115 120 125His
Gln Gln Gly Thr Ala Leu Val Gln His Ile Gly Val Asn Glu Leu 130 135
140Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu
Gly145 150 155 160Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe
Val Thr Asp Arg 165 170 175Gly Val Asp Gly Leu Asp Ile Asp Ile Glu
His Glu Phe Thr Asn Lys 180 185 190Arg Thr Pro Glu Glu Asp Ala Arg
Ala Leu Asn Val Phe Lys Glu Ile 195 200 205Ala Gln Leu Ile Gly Lys
Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile 210 215 220Met Asp Thr Thr
Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile225 230 235 240Ala
Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly 245 250
255Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe
Phe Glu 275 280 285Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val
Asn Glu Tyr Asp 290 295 300Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile
Glu Gly Thr Arg Ala Lys305 310 315 320Lys Tyr Ala Glu Trp Gln Pro
Ser Thr Gly Gly Leu Lys Ala Gly Ile 325 330 335Phe Ser Tyr Ala Ile
Asp Arg Asp Gly Val Ala His Val Pro Ser Thr 340 345 350Tyr Lys Asn
Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn 355 360 365Ile
Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met 370 375
380Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro
Asp385 390 395 400Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly
Gln Tyr Lys Gly 405 410 415Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val
Leu Thr Gly Asp Lys Ile 420 425 430Gln Asn Leu Lys Gly Leu Glu Lys
Leu Ser Lys Leu Gln Lys Leu Glu 435 440 445Leu Arg Gln Leu Ser Asn
Val Lys Glu Ile Thr Pro Glu Leu Leu Pro 450 455 460Glu Ser Met Lys
Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly465 470 475 480Leu
Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly 485 490
495Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu
Leu Met 515 520 525Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile
Thr Val Lys Asn 530 535 540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly
Tyr Tyr Pro Gln Thr Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr
Asp Val Asp Asn Ala Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val
Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu
Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg
Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615
620Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu
Thr625 630
635 640Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val
Asp 645 650 655Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu
Asn Ile Gly 660 665 670Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys
Gly Ala Lys Val Ile 675 680 685Gly Thr Ser Gly Asp Phe Glu Gln Ala
Lys Lys Ile Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg Phe Phe Thr
Trp Gly Gln Thr Asn Trp Ile Ala Phe705 710 715 720Asp Leu Gly Glu
Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730 735Glu Thr
Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys 740 745
750Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn
Trp Thr 770 775 780Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe
Asn Ser Lys Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr Trp Arg
Phe Cys Val Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro Gln Tyr
Thr Glu Leu Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn Asp Val
Ala Asn Thr Leu Asp 835 84010842PRTArtificial SequenceSynthetic
10Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala1
5 10 15Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu
Asn 20 25 30Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr
Asp Gln 35 40 45Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly
Lys Arg Gly 50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp
Arg Ala Ser Thr65 70 75 80Gly Ile Asp Gly Lys Gln Gln His Pro Glu
Asn Thr Met Ala Glu Val 85 90 95Pro Lys Glu Val Asp Ile Leu Phe Val
Phe His Asp His Thr Ala Ser 100 105 110Asp Ser Pro Phe Trp Ser Glu
Leu Lys Asp Ser Tyr Val His Lys Leu 115 120 125His Gln Gln Gly Thr
Ala Leu Val Gln Lys Ile Gly Val Asn Glu Leu 130 135 140Asn Gly Arg
Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly145 150 155
160Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175Gly Val Asp Gly Leu Asp Ile Asp Ile Glu His Glu Phe Thr
Asn Lys 180 185 190Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val
Phe Lys Glu Ile 195 200 205Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp
Lys Ser Lys Leu Leu Ile 210 215 220Met Asp Thr Thr Leu Ser Val Glu
Asn Asn Pro Ile Phe Lys Gly Ile225 230 235 240Ala Glu Asp Leu Asp
Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly 245 250 255Gly Glu Ala
Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln 260 265 270Asn
Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu 275 280
285Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg
Ala Lys305 310 315 320Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly
Leu Lys Ala Gly Ile 325 330 335Phe Ser Tyr Ala Ile Asp Arg Asp Gly
Val Ala His Val Pro Ser Thr 340 345 350Tyr Lys Asn Arg Thr Ser Thr
Asn Leu Gln Arg His Glu Val Asp Asn 355 360 365Ile Ser His Thr Asp
Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met 370 375 380Thr Glu Asp
Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp385 390 395
400Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp
Lys Ile 420 425 430Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu
Gln Lys Leu Glu 435 440 445Leu Arg Gln Leu Ser Asn Val Lys Glu Ile
Thr Pro Glu Leu Leu Pro 450 455 460Glu Ser Met Lys Lys Asp Ala Glu
Leu Val Met Val Gly Met Thr Gly465 470 475 480Leu Glu Lys Leu Asn
Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly 485 490 495Ile Asp Val
Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His 500 505 510Asn
Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met 515 520
525Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln
Thr Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn
Ala Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val Phe Gly Thr Val
Thr Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu Glu Ala Phe Ala
Ile Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg Gln Tyr Val Ser
Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615 620Tyr Lys Gly
Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr625 630 635
640Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn
Ile Gly 660 665 670Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly
Ala Lys Val Ile 675 680 685Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys
Lys Ile Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg Phe Phe Thr Trp
Gly Gln Thr Asn Trp Ile Ala Phe705 710 715 720Asp Leu Gly Glu Ile
Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730 735Glu Thr Asn
Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys 740 745 750Gly
Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met 755 760
765Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser
Lys Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys
Val Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu
Leu Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn Asp Val Ala Asn
Thr Leu Asp 835 84011842PRTArtificial SequenceSynthetic 11Met Asp
Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala1 5 10 15Ala
Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn 20 25
30Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg
Gly 50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala
Ser Thr65 70 75 80Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr
Met Ala Glu Val 85 90 95Pro Lys Glu Val Asp Ile Leu Phe Val Phe His
Asp His Thr Ala Ser 100 105 110Asp Ser Pro Phe Trp Ser Glu Leu Lys
Asp Ser Tyr Val His Lys Leu 115 120 125His Gln Gln Gly Thr Ala Leu
Val Gln Leu Ile Gly Val Asn Glu Leu 130 135 140Asn Gly Arg Thr Gly
Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly145 150 155 160Asn Lys
Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg 165 170
175Gly Val Asp Gly Leu Asp Ile Asp Ile Glu His Glu Phe Thr Asn Lys
180 185 190Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys
Glu Ile 195 200 205Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser
Lys Leu Leu Ile 210 215 220Met Asp Thr Thr Leu Ser Val Glu Asn Asn
Pro Ile Phe Lys Gly Ile225 230 235 240Ala Glu Asp Leu Asp Tyr Leu
Leu Arg Gln Tyr Tyr Gly Ser Gln Gly 245 250 255Gly Glu Ala Glu Val
Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln 260 265 270Asn Tyr Ile
Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu 275 280 285Glu
Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp 290 295
300Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala
Lys305 310 315 320Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu
Lys Ala Gly Ile 325 330 335Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
Ala His Val Pro Ser Thr 340 345 350Tyr Lys Asn Arg Thr Ser Thr Asn
Leu Gln Arg His Glu Val Asp Asn 355 360 365Ile Ser His Thr Asp Tyr
Thr Val Ser Arg Lys Leu Lys Thr Leu Met 370 375 380Thr Glu Asp Lys
Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp385 390 395 400Pro
Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly 405 410
415Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys
Leu Glu 435 440 445Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro
Glu Leu Leu Pro 450 455 460Glu Ser Met Lys Lys Asp Ala Glu Leu Val
Met Val Gly Met Thr Gly465 470 475 480Leu Glu Lys Leu Asn Leu Ser
Gly Leu Asn Arg Gln Thr Leu Asp Gly 485 490 495Ile Asp Val Asn Ser
Ile Thr His Leu Thr Ser Phe Asp Ile Ser His 500 505 510Asn Ser Leu
Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met 515 520 525Thr
Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn 530 535
540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr
Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala
Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val Phe Gly Thr Val Thr
Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu Glu Ala Phe Ala Ile
Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg Gln Tyr Val Ser Lys
Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615 620Tyr Lys Gly Tyr
Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr625 630 635 640Val
Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp 645 650
655Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys
Val Ile 675 680 685Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile
Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln
Thr Asn Trp Ile Ala Phe705 710 715 720Asp Leu Gly Glu Ile Asn Leu
Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730 735Glu Thr Asn Thr Glu
Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys 740 745 750Gly Arg Leu
Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met 755 760 765Asp
Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr 770 775
780Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys
Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val
Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu
Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn Asp Val Ala Asn Thr
Leu Asp 835 84012842PRTArtificial SequenceSynthetic 12Met Asp Lys
His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala1 5 10 15Ala Thr
Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn 20 25 30Thr
Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln 35 40
45Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser
Thr65 70 75 80Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met
Ala Glu Val 85 90 95Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp
His Thr Ala Ser 100 105 110Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp
Ser Tyr Val His Lys Leu 115 120 125His Gln Gln Gly Thr Ala Leu Val
Gln Met Ile Gly Val Asn Glu Leu 130 135 140Asn Gly Arg Thr Gly Leu
Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly145 150 155 160Asn Lys Ala
Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg 165 170 175Gly
Val Asp Gly Leu Asp Ile Asp Ile Glu His Glu Phe Thr Asn Lys 180 185
190Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu
Leu Ile 210 215 220Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile
Phe Lys Gly Ile225 230 235 240Ala Glu Asp Leu Asp Tyr Leu Leu Arg
Gln Tyr Tyr Gly Ser Gln Gly 245 250 255Gly Glu Ala Glu Val Asp Thr
Ile Asn Ser Asp Trp Asn Gln Tyr Gln 260 265 270Asn Tyr Ile Asp Ala
Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu 275 280 285Glu Ser Ala
Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp 290 295 300Pro
Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys305 310
315 320Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly
Ile 325 330 335Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val
Pro Ser Thr 340 345 350Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg
His Glu Val Asp Asn 355 360 365Ile Ser His Thr Asp Tyr Thr Val Ser
Arg Lys Leu Lys Thr Leu Met 370 375 380Thr Glu Asp Lys Arg Tyr Asp
Val Ile Asp Gln Lys Asp Ile Pro Asp385 390 395 400Pro Ala Leu Arg
Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly 405 410 415Asp Leu
Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile 420 425
430Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu
Leu Pro 450 455 460Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val
Gly Met Thr Gly465 470 475 480Leu Glu Lys Leu Asn Leu Ser Gly Leu
Asn Arg Gln Thr Leu Asp Gly 485 490 495Ile Asp Val Asn Ser Ile Thr
His Leu Thr Ser Phe Asp Ile Ser His 500 505 510Asn Ser Leu Asp Leu
Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met 515 520 525Thr Leu Met
Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn 530 535 540Thr
Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr545 550
555 560Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His
Asp
Ile 565 570 575Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn
Thr Phe Ile 580 585 590Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu
Gly Ala Val Asp Gly 595 600 605Arg Gln Tyr Val Ser Lys Asp Tyr Thr
Tyr Glu Ala Phe Arg Lys Asp 610 615 620Tyr Lys Gly Tyr Lys Val His
Leu Thr Ala Ser Asn Leu Gly Glu Thr625 630 635 640Val Thr Ser Lys
Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp 645 650 655Val Ser
Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly 660 665
670Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp
Gly Glu 690 695 700Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn
Trp Ile Ala Phe705 710 715 720Asp Leu Gly Glu Ile Asn Leu Ala Lys
Glu Trp Arg Leu Phe Asn Ala 725 730 735Glu Thr Asn Thr Glu Ile Lys
Thr Asp Ser Ser Leu Asn Val Ala Lys 740 745 750Gly Arg Leu Gln Ile
Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met 755 760 765Asp Ile Lys
Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr 770 775 780Asp
Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu785 790
795 800Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly
Ala 805 810 815Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu
Gly Gln Arg 820 825 830Leu Ser Asn Asp Val Ala Asn Thr Leu Asp 835
84013842PRTArtificial SequenceSynthetic 13Met Asp Lys His Leu Leu
Val Lys Arg Thr Leu Gly Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly
Ala Ala Leu Ala Thr His His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala
Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly
Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu
Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr65 70 75
80Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala
Ser 100 105 110Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val
His Lys Leu 115 120 125His Gln Gln Gly Thr Ala Leu Val Gln Asn Ile
Gly Val Asn Glu Leu 130 135 140Asn Gly Arg Thr Gly Leu Ser Lys Asp
Tyr Pro Asp Thr Pro Glu Gly145 150 155 160Asn Lys Ala Leu Ala Ala
Ala Ile Val Lys Ala Phe Val Thr Asp Arg 165 170 175Gly Val Asp Gly
Leu Asp Ile Asp Ile Glu His Glu Phe Thr Asn Lys 180 185 190Arg Thr
Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile 195 200
205Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys
Gly Ile225 230 235 240Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr
Tyr Gly Ser Gln Gly 245 250 255Gly Glu Ala Glu Val Asp Thr Ile Asn
Ser Asp Trp Asn Gln Tyr Gln 260 265 270Asn Tyr Ile Asp Ala Ser Gln
Phe Met Ile Gly Phe Ser Phe Phe Glu 275 280 285Glu Ser Ala Ser Lys
Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp 290 295 300Pro Asn Asn
Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys305 310 315
320Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro
Ser Thr 340 345 350Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His
Glu Val Asp Asn 355 360 365Ile Ser His Thr Asp Tyr Thr Val Ser Arg
Lys Leu Lys Thr Leu Met 370 375 380Thr Glu Asp Lys Arg Tyr Asp Val
Ile Asp Gln Lys Asp Ile Pro Asp385 390 395 400Pro Ala Leu Arg Glu
Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly 405 410 415Asp Leu Glu
Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile 420 425 430Gln
Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu 435 440
445Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met
Thr Gly465 470 475 480Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg
Gln Thr Leu Asp Gly 485 490 495Ile Asp Val Asn Ser Ile Thr His Leu
Thr Ser Phe Asp Ile Ser His 500 505 510Asn Ser Leu Asp Leu Ser Glu
Lys Ser Glu Asp Arg Lys Leu Leu Met 515 520 525Thr Leu Met Glu Gln
Val Ser Asn His Gln Lys Ile Thr Val Lys Asn 530 535 540Thr Ala Phe
Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr545 550 555
560Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr
Phe Ile 580 585 590Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly
Ala Val Asp Gly 595 600 605Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr
Glu Ala Phe Arg Lys Asp 610 615 620Tyr Lys Gly Tyr Lys Val His Leu
Thr Ala Ser Asn Leu Gly Glu Thr625 630 635 640Val Thr Ser Lys Val
Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp 645 650 655Val Ser Asp
Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly 660 665 670Ser
Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile 675 680
685Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile
Ala Phe705 710 715 720Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp
Arg Leu Phe Asn Ala 725 730 735Glu Thr Asn Thr Glu Ile Lys Thr Asp
Ser Ser Leu Asn Val Ala Lys 740 745 750Gly Arg Leu Gln Ile Leu Lys
Asp Thr Thr Ile Asp Leu Glu Lys Met 755 760 765Asp Ile Lys Asn Arg
Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr 770 775 780Asp Val Ala
Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu785 790 795
800Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly
Gln Arg 820 825 830Leu Ser Asn Asp Val Ala Asn Thr Leu Asp 835
84014842PRTArtificial SequenceSynthetic 14Met Asp Lys His Leu Leu
Val Lys Arg Thr Leu Gly Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly
Ala Ala Leu Ala Thr His His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala
Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly
Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu
Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr65 70 75
80Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala
Ser 100 105 110Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val
His Lys Leu 115 120 125His Gln Gln Gly Thr Ala Leu Val Gln Gln Ile
Gly Val Asn Glu Leu 130 135 140Asn Gly Arg Thr Gly Leu Ser Lys Asp
Tyr Pro Asp Thr Pro Glu Gly145 150 155 160Asn Lys Ala Leu Ala Ala
Ala Ile Val Lys Ala Phe Val Thr Asp Arg 165 170 175Gly Val Asp Gly
Leu Asp Ile Asp Ile Glu His Glu Phe Thr Asn Lys 180 185 190Arg Thr
Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile 195 200
205Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys
Gly Ile225 230 235 240Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr
Tyr Gly Ser Gln Gly 245 250 255Gly Glu Ala Glu Val Asp Thr Ile Asn
Ser Asp Trp Asn Gln Tyr Gln 260 265 270Asn Tyr Ile Asp Ala Ser Gln
Phe Met Ile Gly Phe Ser Phe Phe Glu 275 280 285Glu Ser Ala Ser Lys
Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp 290 295 300Pro Asn Asn
Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys305 310 315
320Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro
Ser Thr 340 345 350Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His
Glu Val Asp Asn 355 360 365Ile Ser His Thr Asp Tyr Thr Val Ser Arg
Lys Leu Lys Thr Leu Met 370 375 380Thr Glu Asp Lys Arg Tyr Asp Val
Ile Asp Gln Lys Asp Ile Pro Asp385 390 395 400Pro Ala Leu Arg Glu
Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly 405 410 415Asp Leu Glu
Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile 420 425 430Gln
Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu 435 440
445Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met
Thr Gly465 470 475 480Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg
Gln Thr Leu Asp Gly 485 490 495Ile Asp Val Asn Ser Ile Thr His Leu
Thr Ser Phe Asp Ile Ser His 500 505 510Asn Ser Leu Asp Leu Ser Glu
Lys Ser Glu Asp Arg Lys Leu Leu Met 515 520 525Thr Leu Met Glu Gln
Val Ser Asn His Gln Lys Ile Thr Val Lys Asn 530 535 540Thr Ala Phe
Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr545 550 555
560Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr
Phe Ile 580 585 590Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly
Ala Val Asp Gly 595 600 605Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr
Glu Ala Phe Arg Lys Asp 610 615 620Tyr Lys Gly Tyr Lys Val His Leu
Thr Ala Ser Asn Leu Gly Glu Thr625 630 635 640Val Thr Ser Lys Val
Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp 645 650 655Val Ser Asp
Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly 660 665 670Ser
Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile 675 680
685Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile
Ala Phe705 710 715 720Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp
Arg Leu Phe Asn Ala 725 730 735Glu Thr Asn Thr Glu Ile Lys Thr Asp
Ser Ser Leu Asn Val Ala Lys 740 745 750Gly Arg Leu Gln Ile Leu Lys
Asp Thr Thr Ile Asp Leu Glu Lys Met 755 760 765Asp Ile Lys Asn Arg
Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr 770 775 780Asp Val Ala
Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu785 790 795
800Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly
Gln Arg 820 825 830Leu Ser Asn Asp Val Ala Asn Thr Leu Asp 835
84015842PRTArtificial SequenceSynthetic 15Met Asp Lys His Leu Leu
Val Lys Arg Thr Leu Gly Cys Val Cys Ala1 5 10 15Ala Thr Leu Met Gly
Ala Ala Leu Ala Thr His His Asp Ser Leu Asn 20 25 30Thr Val Lys Ala
Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln 35 40 45Gln Val Gly
Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly 50 55 60Pro Leu
Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr65 70 75
80Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala
Ser 100 105 110Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val
His Lys Leu 115 120 125His Gln Gln Gly Thr Ala Leu Val Gln Arg Ile
Gly Val Asn Glu Leu 130 135 140Asn Gly Arg Thr Gly Leu Ser Lys Asp
Tyr Pro Asp Thr Pro Glu Gly145 150 155 160Asn Lys Ala Leu Ala Ala
Ala Ile Val Lys Ala Phe Val Thr Asp Arg 165 170 175Gly Val Asp Gly
Leu Asp Ile Asp Ile Glu His Glu Phe Thr Asn Lys 180 185 190Arg Thr
Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile 195 200
205Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys
Gly Ile225 230 235 240Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr
Tyr Gly Ser Gln Gly 245 250 255Gly Glu Ala Glu Val Asp Thr Ile Asn
Ser Asp Trp Asn Gln Tyr Gln 260 265 270Asn Tyr Ile Asp Ala Ser Gln
Phe Met Ile Gly Phe Ser Phe Phe Glu 275 280 285Glu Ser Ala Ser Lys
Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp 290 295 300Pro Asn Asn
Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys305 310 315
320Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro
Ser Thr 340 345 350Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His
Glu Val Asp Asn 355 360 365Ile Ser His Thr Asp Tyr Thr Val Ser Arg
Lys Leu Lys Thr Leu Met 370 375 380Thr Glu Asp Lys Arg Tyr Asp Val
Ile Asp Gln Lys Asp Ile Pro Asp385 390 395 400Pro Ala Leu Arg Glu
Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly 405 410 415Asp Leu Glu
Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile 420 425 430Gln
Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu 435 440
445Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met
Thr Gly465 470 475 480Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg
Gln Thr Leu Asp Gly 485 490 495Ile Asp Val Asn
Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His 500 505 510Asn Ser
Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met 515 520
525Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln
Thr Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn
Ala Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val Phe Gly Thr Val
Thr Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu Glu Ala Phe Ala
Ile Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg Gln Tyr Val Ser
Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615 620Tyr Lys Gly
Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr625 630 635
640Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn
Ile Gly 660 665 670Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly
Ala Lys Val Ile 675 680 685Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys
Lys Ile Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg Phe Phe Thr Trp
Gly Gln Thr Asn Trp Ile Ala Phe705 710 715 720Asp Leu Gly Glu Ile
Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730 735Glu Thr Asn
Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys 740 745 750Gly
Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met 755 760
765Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser
Lys Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys
Val Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu
Leu Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn Asp Val Ala Asn
Thr Leu Asp 835 84016842PRTArtificial SequenceSynthetic 16Met Asp
Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala1 5 10 15Ala
Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn 20 25
30Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg
Gly 50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala
Ser Thr65 70 75 80Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr
Met Ala Glu Val 85 90 95Pro Lys Glu Val Asp Ile Leu Phe Val Phe His
Asp His Thr Ala Ser 100 105 110Asp Ser Pro Phe Trp Ser Glu Leu Lys
Asp Ser Tyr Val His Lys Leu 115 120 125His Gln Gln Gly Thr Ala Leu
Val Gln Val Ile Gly Val Asn Glu Leu 130 135 140Asn Gly Arg Thr Gly
Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly145 150 155 160Asn Lys
Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg 165 170
175Gly Val Asp Gly Leu Asp Ile Asp Ile Glu His Glu Phe Thr Asn Lys
180 185 190Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys
Glu Ile 195 200 205Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser
Lys Leu Leu Ile 210 215 220Met Asp Thr Thr Leu Ser Val Glu Asn Asn
Pro Ile Phe Lys Gly Ile225 230 235 240Ala Glu Asp Leu Asp Tyr Leu
Leu Arg Gln Tyr Tyr Gly Ser Gln Gly 245 250 255Gly Glu Ala Glu Val
Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln 260 265 270Asn Tyr Ile
Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu 275 280 285Glu
Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp 290 295
300Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala
Lys305 310 315 320Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu
Lys Ala Gly Ile 325 330 335Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val
Ala His Val Pro Ser Thr 340 345 350Tyr Lys Asn Arg Thr Ser Thr Asn
Leu Gln Arg His Glu Val Asp Asn 355 360 365Ile Ser His Thr Asp Tyr
Thr Val Ser Arg Lys Leu Lys Thr Leu Met 370 375 380Thr Glu Asp Lys
Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp385 390 395 400Pro
Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly 405 410
415Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys
Leu Glu 435 440 445Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro
Glu Leu Leu Pro 450 455 460Glu Ser Met Lys Lys Asp Ala Glu Leu Val
Met Val Gly Met Thr Gly465 470 475 480Leu Glu Lys Leu Asn Leu Ser
Gly Leu Asn Arg Gln Thr Leu Asp Gly 485 490 495Ile Asp Val Asn Ser
Ile Thr His Leu Thr Ser Phe Asp Ile Ser His 500 505 510Asn Ser Leu
Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met 515 520 525Thr
Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn 530 535
540Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr
Tyr545 550 555 560Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala
Glu His Asp Ile 565 570 575Leu Thr Asp Phe Val Phe Gly Thr Val Thr
Lys Arg Asn Thr Phe Ile 580 585 590Gly Asp Glu Glu Ala Phe Ala Ile
Tyr Lys Glu Gly Ala Val Asp Gly 595 600 605Arg Gln Tyr Val Ser Lys
Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp 610 615 620Tyr Lys Gly Tyr
Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr625 630 635 640Val
Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp 645 650
655Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys
Val Ile 675 680 685Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile
Phe Asp Gly Glu 690 695 700Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln
Thr Asn Trp Ile Ala Phe705 710 715 720Asp Leu Gly Glu Ile Asn Leu
Ala Lys Glu Trp Arg Leu Phe Asn Ala 725 730 735Glu Thr Asn Thr Glu
Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys 740 745 750Gly Arg Leu
Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met 755 760 765Asp
Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr 770 775
780Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys
Leu785 790 795 800Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val
Asp Gly Gly Ala 805 810 815Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu
Gln Ile Leu Gly Gln Arg 820 825 830Leu Ser Asn Asp Val Ala Asn Thr
Leu Asp 835 84017842PRTArtificial SequenceSynthetic 17Met Asp Lys
His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala1 5 10 15Ala Thr
Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn 20 25 30Thr
Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln 35 40
45Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser
Thr65 70 75 80Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met
Ala Glu Val 85 90 95Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp
His Thr Ala Ser 100 105 110Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp
Ser Tyr Val His Lys Leu 115 120 125His Gln Gln Gly Thr Ala Leu Val
Gln Trp Ile Gly Val Asn Glu Leu 130 135 140Asn Gly Arg Thr Gly Leu
Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly145 150 155 160Asn Lys Ala
Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg 165 170 175Gly
Val Asp Gly Leu Asp Ile Asp Ile Glu His Glu Phe Thr Asn Lys 180 185
190Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu
Leu Ile 210 215 220Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile
Phe Lys Gly Ile225 230 235 240Ala Glu Asp Leu Asp Tyr Leu Leu Arg
Gln Tyr Tyr Gly Ser Gln Gly 245 250 255Gly Glu Ala Glu Val Asp Thr
Ile Asn Ser Asp Trp Asn Gln Tyr Gln 260 265 270Asn Tyr Ile Asp Ala
Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu 275 280 285Glu Ser Ala
Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp 290 295 300Pro
Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys305 310
315 320Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly
Ile 325 330 335Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val
Pro Ser Thr 340 345 350Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg
His Glu Val Asp Asn 355 360 365Ile Ser His Thr Asp Tyr Thr Val Ser
Arg Lys Leu Lys Thr Leu Met 370 375 380Thr Glu Asp Lys Arg Tyr Asp
Val Ile Asp Gln Lys Asp Ile Pro Asp385 390 395 400Pro Ala Leu Arg
Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly 405 410 415Asp Leu
Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile 420 425
430Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu
Leu Pro 450 455 460Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val
Gly Met Thr Gly465 470 475 480Leu Glu Lys Leu Asn Leu Ser Gly Leu
Asn Arg Gln Thr Leu Asp Gly 485 490 495Ile Asp Val Asn Ser Ile Thr
His Leu Thr Ser Phe Asp Ile Ser His 500 505 510Asn Ser Leu Asp Leu
Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met 515 520 525Thr Leu Met
Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn 530 535 540Thr
Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr545 550
555 560Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp
Ile 565 570 575Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn
Thr Phe Ile 580 585 590Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu
Gly Ala Val Asp Gly 595 600 605Arg Gln Tyr Val Ser Lys Asp Tyr Thr
Tyr Glu Ala Phe Arg Lys Asp 610 615 620Tyr Lys Gly Tyr Lys Val His
Leu Thr Ala Ser Asn Leu Gly Glu Thr625 630 635 640Val Thr Ser Lys
Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp 645 650 655Val Ser
Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly 660 665
670Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp
Gly Glu 690 695 700Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn
Trp Ile Ala Phe705 710 715 720Asp Leu Gly Glu Ile Asn Leu Ala Lys
Glu Trp Arg Leu Phe Asn Ala 725 730 735Glu Thr Asn Thr Glu Ile Lys
Thr Asp Ser Ser Leu Asn Val Ala Lys 740 745 750Gly Arg Leu Gln Ile
Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met 755 760 765Asp Ile Lys
Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr 770 775 780Asp
Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu785 790
795 800Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly
Ala 805 810 815Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu
Gly Gln Arg 820 825 830Leu Ser Asn Asp Val Ala Asn Thr Leu Asp 835
84018451PRTArtificial SequenceSynthetic 18Gln Val Gln Leu Gln Gln
Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp
Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile
Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp
Gly 100 105 110Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420
425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 435 440 445Pro Gly Lys 45019212PRTArtificial SequenceSynthetic
19Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly1
5 10 15Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr
Ile 20 25 30His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp
Ile Tyr 35 40 45Ala Thr Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser
Gly Ser Gly 50 55 60Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val
Glu Ala Glu Asp65 70 75 80Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr
Ser Asn Pro Pro Thr Phe 85 90 95Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg Thr Val Ala Ala Pro Ser 100 105 110Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly Thr Ala 115 120 125Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser145 150 155
160Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
165 170 175Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala Cys 180 185 190Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser Phe Asn 195 200 205Arg Gly Glu Cys 210
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References